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Friday, February 28th, 2014

Davis, S. M., Thomas, A. L., Nomie, K. J., Huang, L. and Dierick, H. A. (2014). Tailless and Atrophin control Drosophila aggression by regulating neuropeptide signalling in the pars intercerebralis. Nat Commun 5: 3177. PubMed ID: 24495972
Summary: Aggressive behaviour is widespread throughout the animal kingdom. However, its mechanisms are poorly understood, and the degree of molecular conservation between distantly related species is unknown. This study shows that knockdown of tailless (tll) increases aggression in Drosophila, similar to the effect of its mouse orthologue Nr2e1. Tll localizes to the adult pars intercerebralis (PI), which shows similarity to the mammalian hypothalamus. Knockdown of tll in the PI is sufficient to increase aggression and is rescued by co-expressing human NR2E1. Knockdown of Atrophin, a Tll co-repressor, also increases aggression, and both proteins physically interact in the PI. tll knockdown-induced aggression is fully suppressed by blocking neuropeptide processing or release from the PI. In addition, genetically activating PI neurons increases aggression, mimicking the aggression-inducing effect of hypothalamic stimulation. Together, these results suggest that a transcriptional control module regulates neuropeptide signalling from the neurosecretory cells of the brain to control aggressive behaviour.

Hutchinson, K. M., Vonhoff, F. and Duch, C. (2014). Dscam1 is required for normal dendrite growth and branching but not for dendritic spacing in Drosophila motoneurons. J Neurosci 34: 1924-1931. PubMed ID: 24478371
Summary: Down syndrome cell adhesion molecule, Dscam, serves diverse neurodevelopmental functions, including axon guidance and synaptic adhesion, as well as self-recognition and self-avoidance, depending on the neuron type, brain region, or species under investigation. In Drosophila, the extensive molecular diversity that results from alternative splicing of Dscam1 into >38,000 isoforms provides neurons with a unique molecular code for self-recognition in the nervous system. Each neuron produces only a small subset of Dscam1 isoforms, and distinct Dscam1 isoforms mediate homophilic interactions, which in turn, result in repulsion and even spacing of self-processes, while allowing contact with neighboring cells. While these mechanisms have been shown to underlie mushroom body development and spacing of mechanosensory neuron dendrites, this study reports that Dscam1 plays no role in adult Drosophila motoneuron dendrite spacing, but is required for motoneuron dendritic growth. Targeted expression of Dscam-RNAi in an identified flight motoneuron did not impact dendrite spacing, but instead produced overgrowth. Increasing the knockdown strength severely reduced dendritic growth and branching. Similarly, Dscam mutant motoneurons in an otherwise control background (MARCM) were completely devoid of mature dendrites. These data suggest that Dscam1 is required cell autonomously for normal adult motoneuron dendrite growth in Drosophila. This demonstrates a previously unreported role of Drosophila Dscam1 in central neuron development, and expands the current understanding that Dscam1 operates as a cell adhesion molecule that mediates homophilic repulsion.

White-Grindley, E., Li, L., Mohammad Khan, R., Ren, F., Saraf, A., Florens, L. and Si, K. (2014). Contribution of Orb2A Stability in Regulated Amyloid-Like Oligomerization of Drosophila Orb2. PLoS Biol 12: e1001786. PubMed ID: 24523662
Summary: How learned experiences persist as memory for a long time is an important question. In Drosophila the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. This study reports the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

Hoerndli, F. J., Maxfield, D. A., Brockie, P. J., Mellem, J. E., Jensen, E., Wang, R., Madsen, D. M. and Maricq, A. V. (2013). Kinesin-1 regulates synaptic strength by mediating the delivery, removal, and redistribution of AMPA receptors. Neuron 80: 14237. PubMed ID: 24360545
Summary: A primary determinant of the strength of neurotransmission is the number of AMPA-type glutamate receptors (AMPARs; see Drosophila Glutamate receptor IIA) at synapses. However, a mechanistic understanding of how the number of synaptic AMPARs is regulated is lacking. This study shows that UNC-116 (see Drosophila Zipper), the C. elegans homolog of vertebrate kinesin-1 heavy chain (KIF5), modifies synaptic strength by mediating the rapid delivery, removal, and redistribution of synaptic AMPARs. Furthermore, by studying the real-time transport of C. elegans AMPAR subunits in vivo, it was demonstrated that although homomeric GLR-1 AMPARs can diffuse to and accumulate at synapses in unc-116 mutants, glutamate-gated currents are diminished because heteromeric GLR-1/GLR-2 receptors do not reach synapses in the absence of UNC-116/KIF5-mediated transport. These data support a model in which ongoing motor-driven delivery and removal of AMPARs controls not only the number but also the composition of synaptic AMPARs, and thus the strength of synaptic transmission.

Thursday, February 27th

Yu, S., Waldholm, J., Bohm, S. and Visa, N. (2014). Brahma regulates a specific trans-splicing event at the mod(mdg4) locus of Drosophila melanogaster. RNA Biol 11 [Epub ahead of print]. PubMed ID: 24526065
Summary: The mod(mdg4) locus of Drosophila contains several transcription units encoded on both DNA strands. The mod(mdg4) pre-mRNAs are alternatively spliced, and a very significant fraction of the mature mod(mdg4) mRNAs are formed by trans-splicing. The transcripts derived from one of the anti-sense regions within the mod(mdg4) locus was studied in order to shed light on the expression of this complex locus. The expression of anti-sense mod(mdg4) transcripts was characterized in S2 cells, their transcription start sites and cleavage sites mapped, alternatively spliced transcripts were identified and quantified, and insight was obtained into the regulation of the mod(mdg4) trans-splicing. In a previous study, it was shown that the alternative splicing of some mod(mdg4) transcripts was regulated by Brahma (BRM), the ATPase subunit of the SWI/SNF chromatin-remodeling complex. This study shows, using RNA interference and overexpression of recombinant BRM proteins, that the levels of BRM affect specifically the abundance of a trans-spliced mod(mdg4) mRNA isoform in both S2 cells and larvae. This specific effect on trans-splicing is accompanied by a local increase in the density of RNA polymerase II and by a change in the phosphorylation state of the C-terminal domain of the large subunit of RNA polymerase II. Interestingly, the regulation of the mod(mdg4) splicing by BRM is independent of the ATPase activity of BRM, which suggests that the mechanism by which BRM modulates trans-splicing is independent of its chromatin-remodeling activity.

Shpiz, S., Ryazansky, S., Olovnikov, I., Abramov, Y. and Kalmykova, A. (2014). Euchromatic Transposon Insertions Trigger Production of Novel Pi- and Endo-siRNAs at the Target Sites in the Drosophila Germline. PLoS Genet 10: e1004138. PubMed ID: 24516406
Summary: The control of transposable element (TE) activity in germ cells provides genome integrity over generations. A distinct small RNA-mediated pathway utilizing Piwi-interacting RNAs (piRNAs) suppresses TE expression in gonads of metazoans. In the fly, primary piRNAs derive from so-called piRNA clusters, which are enriched in damaged repeated sequences. These piRNAs launch a cycle of TE and piRNA cluster transcript cleavages resulting in the amplification of piRNA and TE silencing. Using genome-wide comparison of TE insertions and ovarian small RNA libraries from two Drosophila strains, it was found that individual TEs inserted into euchromatic loci form novel dual-stranded piRNA clusters. Formation of the piRNA-generating loci by active individual TEs provides a more potent silencing response to the TE expansion. Like all piRNA clusters, individual TEs are also capable of triggering the production of endogenous small interfering (endo-si) RNAs. Small RNA production by individual TEs spreads into the flanking genomic regions including coding cellular genes. Formation of TE-associated small RNA clusters were shown to be able to down-regulate expression of nearby genes in ovaries. Integration of TEs into the 3' untranslated region of actively transcribed genes induces piRNA production towards the 3'-end of transcripts, causing the appearance of genic piRNA clusters, a phenomenon that has been reported in different organisms. These data suggest a significant role of TE-associated small RNAs in the evolution of regulatory networks in the germline.

Lyu, Y., Shen, Y., Li, H., Chen, Y., Guo, L., Zhao, Y., Hungate, E., Shi, S., Wu, C. I. and Tang, T. (2014). New microRNAs in Drosophila-birth, death and cycles of adaptive evolution. PLoS Genet 10: e1004096. PubMed ID: 24465220
Summary: The origin and evolution of new microRNAs (miRNAs) is important because they can impact the transcriptome broadly. As miRNAs can potentially emerge constantly and rapidly, their rates of birth and evolution have been extensively debated. However, most new miRNAs identified appear not to be biologically significant. After an extensive search, twelve new miRNAs were identified that emerged de novo in Drosophila melanogaster in the last 4 million years (Myrs) and have been evolving adaptively. Unexpectedly, even though they are adaptively evolving at birth, more than 94% of such new miRNAs disappear over time. They provide selective advantages, but only for a transient evolutionary period. After 30 Myrs, all surviving miRNAs make the transition from the adaptive phase of rapid evolution to the conservative phase of slow evolution, apparently becoming integrated into the transcriptional network. During this transition, the expression shifts from being tissue-specific, predominantly in testes and larval brain/gonads/imaginal discs, to a broader distribution in many other tissues. Interestingly, a measurable fraction (20-30%) of these conservatively evolving miRNAs experience 'evolutionary rejuvenation' and begin to evolve rapidly again. These rejuvenated miRNAs then start another cycle of adaptive - conservative evolution. In conclusion, the selective advantages driving evolution of miRNAs are themselves evolving, and sometimes changing direction, which highlights the regulatory roles of miRNAs.

McManus, C. J., Coolon, J. D., Eipper-Mains, J., Wittkopp, P. J. and Graveley, B. R. (2014). Evolution of splicing regulatory networks in Drosophila. Genome [Epub ahead of print]. PubMed ID: 24515119
Summary: The proteome expanding effects of alternative pre-mRNA splicing have had a profound impact on eukaryotic evolution. The events that create this diversity can be placed into four major classes - exon skipping, intron retention, alternative 5' splice sites and alternative 3' splice sites. Although the regulatory mechanisms and evolutionary pressures among alternative splicing classes clearly differ, how these differences affect the evolution of splicing regulation remains poorly characterized. This study used RNA-seq to investigate splicing differences in D. simulans, D. sechellia, and three strains of D. melanogaster. Regulation of exon skipping and tandem alternative 3' splice sites (NAGNAGs) are more divergent than other splicing classes. Splicing regulation is most divergent in frame-preserving events and events in non-coding regions. The contributions of cis- and trans-acting changes in splicing regulatory networks was determined by comparing allele-specific splicing in F1 interspecific hybrids, as differences in allele-specific splicing reflect changes in cis-regulatory element activity. It was found that species-specific differences in intron retention and alternative splice site usage are primarily attributable to changes in cis-regulatory elements (median ~80% cis), while species-specific exon skipping differences are driven by both cis- and trans-regulatory divergence (median ~50% cis). These results help define the mechanisms and constraints influencing splicing regulatory evolution, and show that networks regulating the four major classes of alternative splicing are diverging through different genetic mechanisms. A model is proposed in which differences in regulatory network architecture among classes of alternative splicing affect the evolution of splicing regulation (McManus, 2014).

Wednesday, February 26th

Silbereis, J. C., et al. (2014). Olig1 function is required to repress dlx1/2 and interneuron production in Mammalian brain. Neuron 81: 574-587. PubMed ID: 24507192
Summary: Abnormal GABAergic interneuron density, and imbalance of excitatory versus inhibitory tone, is thought to result in epilepsy, neurodevelopmental disorders, and psychiatric disease. Recent studies indicate that interneuron cortical density is determined primarily by the size of the precursor pool in the embryonic telencephalon. However, factors essential for regulating interneuron allocation from telencephalic multipotent precursors are poorly understood. This study reports that Olig1 (see Drosophila Oli) represses production of GABAergic interneurons throughout the mouse brain. Olig1 deletion in mutant mice results in ectopic expression and upregulation of Dlx1/2 genes (Drosophila homolog Distal-less) in the ventral medial ganglionic eminences and adjacent regions of the septum, resulting in an approximately 30% increase in adult cortical interneuron numbers. Olig1 was shown to directly repress the Dlx1/2 I12b intergenic enhancer, and Dlx1/2 was shown to function genetically downstream of Olig1. These findings establish Olig1 as an essential repressor of Dlx1/2 and interneuron production in developing mammalian brain.

Pasquali, L., et al. (2014). Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants. Nat Genet 46: 136-143. PubMed ID: 24413736
Summary: Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. This study has mapped and examined the function of human islet cis-regulatory networks. Genomic sequences were identified that are targeted by islet transcription factors to drive islet-specific gene activity; most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. Sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. These studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.

Bie, B., Wu, J., Yang, H., Xu, J. J., Brown, D. L. and Naguib, M. (2014). Epigenetic suppression of neuroligin 1 underlies amyloid-induced memory deficiency. Nat Neurosci 17: 223-231. PubMed ID: 24441681
Summary: Amyloid-induced microglial activation and neuroinflammation impair central synapses and memory function, although the mechanism remains unclear. Neuroligin 1 [NLGN1; (see Drosophila Neuroligin-1 & Neuroligin-2)], a postsynaptic protein found in central excitatory synapses, governs excitatory synaptic efficacy and plasticity in the brain. This study found, in rodents, that amyloid fibril-induced neuroinflammation enhances the interaction between histone deacetylase 2 and methyl-CpG-binding protein 2, leading to suppressed histone H3 acetylation and enhanced cytosine methylation in the Nlgn1 promoter region and decreased NLGN1 expression, underlying amyloid-induced memory deficiency. Manipulation of microglia-associated neuroinflammation modulates the epigenetic modification of the Nlgn1 promoter, hippocampal glutamatergic transmission and memory function. These findings link neuroinflammation, synaptic efficacy and memory, thus providing insight into the pathogenesis of amyloid-associated diseases.

Hattori, Y., Usui, T., Satoh, D., Moriyama, S., Shimono, K., Itoh, T., Shirahige, K. and Uemura, T. (2013). Sensory-Neuron Subtype-Specific Transcriptional Programs Controlling Dendrite Morphogenesis: Genome-wide Analysis of Abrupt and Knot/Collier. Dev Cell 27: 530-544. PubMed ID: 24290980
Summary: The transcription factors Abrupt (Ab) and Knot (Kn) act as selectors of distinct dendritic arbor morphologies in two classes of Drosophila sensory neurons, termed class I and class IV, respectively. This study performed binding-site mapping and transcriptional profiling of these isolated neurons. Their profiles were similarly enriched in cell-type-specific enhancers of genes implicated in neural development. A total of 429 target genes were identified, of which 56 were common to Ab and Kn; these targets included genes necessary to shape dendritic arbors in either or both of the two sensory subtypes. Furthermore, a common target gene, encoding the cell adhesion molecule Ten-m, was expressed more strongly in class I than class IV, and this differential was critical to the class-selective directional control of dendritic branch sprouting or extension. This analysis illustrates how differentiating neurons employ distinct and shared repertoires of gene expression to produce class-selective morphological traits.

Tuesday, February 25th

Charng, W. L., Yamamoto, S., Jaiswal, M., Bayat, V., Xiong, B., Zhang, K., Sandoval, H., David, G., Gibbs, S., Lu, H. C., Chen, K., Giagtzoglou, N. and Bellen, H. J. (2014). Drosophila Tempura, a Novel Protein Prenyltransferase alpha Subunit, Regulates Notch Signaling Via Rab1 and Rab11. PLoS Biol 12: e1001777. PubMed ID: 24492843
Summary: Vesicular trafficking plays a key role in tuning the activity of Notch signaling. This study describes a novel and conserved Rab geranylgeranyltransferase (RabGGT)-alpha-like subunit that is required for Notch signaling-mediated lateral inhibition and cell fate determination of external sensory organs. This protein is encoded by tempura, and its loss affects the secretion of Scabrous and Delta, two proteins required for proper Notch signaling. Tempura forms a heretofore uncharacterized RabGGT complex that geranylgeranylates Rab1 and Rab11. This geranylgeranylation is required for their proper subcellular localization. A partial dysfunction of Rab1 affects Scabrous and Delta in the secretory pathway. In addition, a partial loss Rab11 affects trafficking of Delta. In summary, Tempura functions as a new geranylgeranyltransferase that regulates the subcellular localization of Rab1 and Rab11, which in turn regulate trafficking of Scabrous and Delta, thereby affecting Notch signaling.

Schafer, G., Narasimha, M., Vogelsang, E. and Leptin, M. (2014). Cadherin switching during the formation and differentiation of the Drosophila mesoderm: implications for epithelial mesenchymal transitions. J Cell Sci [Epub ahead of print]. PubMed ID: 24496448
Summary: Epithelial-to-mesenchymal transitions (EMT) are typically accompanied by downregulation of epithelial (E-) cadherin, and often additionally by upregulation of a mesenchymal/neuronal (N-) cadherin. Snail represses transcription of the E-cadherin gene both during normal development and during tumor spreading. The formation of the mesodermal germ layer in Drosophila, considered a paradigm of a developmental EMT, is associated with Snail-mediated repression of E-cadherin and the upregulation of N-cadherin. Using genetic manipulations to remove or overexpress the cadherins, this study shows that the complementarity of cadherin expression is not necessary for the segregation or the dispersal of the mesodermal germ layer in Drosophila. However differential effects were discovered on E- and N-cadherin on the differentiation of subsets of mesodermal derivatives, which depend on Wingless signalling from the ectoderm, indicating differential abilities of E- and N-cadherin to bind and sequester the common junctional and signalling effector beta-catenin. They suggest that the need to downregulate E-cadherin in the mesoderm may be to facilitate optimal levels of Wingless signalling.

Yatsenko, A. S. and Shcherbata, H. R. (2014). Drosophila miR-9a Targets the ECM Receptor Dystroglycan to Canalize Myotendinous Junction Formation. Dev Cell 28: 335-348. PubMed ID: 24525189
Summary: Establishment of intercellular interactions between various cell types of different origin is vital for organism development and tissue maintenance. Therefore, precise timing, expression pattern, and amounts of extracellular matrix (ECM) proteins must be tightly regulated. Particularly, the ECM is important for the development and function of myotendinous junctions (MTJs). Precise levels of the ECM receptor Dystroglycan (Dg) were found to be required for MTJ formation in Drosophila and that Dg levels in this process are controlled by miR-9a. In the embryo, Dg is enriched at the termini of the growing muscles facing the tendon matrix and absent from miR-9a-expressing tendons. This gradient of Dg expression is crucial for proper muscle-tendon attachments and is adjusted by miR-9a. In addition to Dg, miR-9a regulates the expression of several other critical muscle genes, and we therefore propose that during embryogenesis, miR-9a specifically controls the expression of mesodermal genes to canalize MTJ morphogenesis.

Muliyil, S. and Narasimha, M. (2014). Mitochondrial ROS Regulates Cytoskeletal and Mitochondrial Remodeling to Tune Cell and Tissue Dynamics in a Model for Wound Healing. Dev Cell [Epub ahead of print]. PubMed ID: 24486154
Summary: How cues that trigger the wound response result in tissue healing is a question of immense biological and medical importance. This study uncovers roles for mitochondrial reactive oxygen species (mtROS) during Drosophila dorsal closure, a model for wound healing. By using real-time visualization of ROS activity and single-cell perturbation strategies, it was demonstrated that stochasticities in ROS generation in the amnioserosa are necessary and sufficient to trigger cell delamination. Dose-dependent effects of mtROS were demonstrated on actomyosin and mitochondrial architecture, dynamics, and activity that mediate both stochasticities in cell behavior and the phases of tissue dynamics accompanying dorsal closure. The results establish that ROS levels tune cell behavior and tissue dynamics qualitatively and quantitatively. They identify a pathway triggered by ROS and mediated by the Rho effector ROCK and its substrates that influences tissue patterning and homeostasis through the coordinate regulation of both mitochondrial morphology and tissue tension.

Monday, February 24th

Ahmad, S. M., Busser, B. W., Huang, D., Cozart, E. J., Michaud, S., Zhu, X., Jeffries, N., Aboukhalil, A., Bulyk, M. L., Ovcharenko, I. and Michelson, A. M. (2014). Machine learning classification of cell-specific cardiac enhancers uncovers developmental subnetworks regulating progenitor cell division and cell fate specification. Development 141: 878-888. PubMed ID: 24496624
Summary: The Drosophila heart is composed of two distinct cell types, the contractile cardial cells (CCs) and the surrounding non-muscle pericardial cells (PCs), development of which is regulated by a network of conserved signaling molecules and transcription factors (TFs). This study used machine learning with array-based chromatin immunoprecipitation (ChIP) data and TF sequence motifs to computationally classify cell type-specific cardiac enhancers. Extensive testing of predicted enhancers at single-cell resolution revealed the added value of ChIP data for modeling cell type-specific activities. Furthermore, clustering the top-scoring classifier sequence features identified novel cardiac and cell type-specific regulatory motifs. For example, it was found that the Myb motif learned by the classifier is crucial for CC activity, and the Myb TF acts in concert with two forkhead domain TFs, Jumeau and Checkpoint suppressor homologue (CHES-1-like), and with Polo kinase to regulate cardiac progenitor cell divisions. In addition, differential motif enrichment and cis-trans genetic studies revealed that the Notch signaling pathway TF Suppressor of Hairless [Su(H)] discriminates PC from CC enhancer activities. Collectively, these studies elucidate molecular pathways used in the regulatory decisions for proliferation and differentiation of cardiac progenitor cells, implicate Su(H) in regulating cell fate decisions of these progenitors, and document the utility of enhancer modeling in uncovering developmental regulatory subnetworks.

Wolfram, V., Southall, T. D., Gunay, C., Prinz, A. A., Brand, A. H. and Baines, R. A. (2014). The transcription factors islet and lim3 combinatorially regulate ion channel gene expression. J Neurosci 34: 2538-2543. PubMed ID: 24523544
Summary: Expression of appropriate ion channels is essential to allow developing neurons to form functional networks. Previous studies have identified LIM-homeodomain (HD) transcription factors (TFs), expressed by developing neurons, that are specifically able to regulate ion channel gene expression. This study used the technique of DNA adenine methyltransferase identification (DamID) to identify putative gene targets of four such TFs that are differentially expressed in Drosophila motoneurons. Analysis of targets for Islet (Isl), Lim3, Hb9/ExEx, and Even-skipped (Eve) identifies both ion channel genes and genes predicted to regulate aspects of dendritic and axonal morphology. Significantly, some ion channel genes are bound by more than one TF, consistent with the possibility of combinatorial regulation. One such gene is Shaker (Sh), which encodes a voltage-dependent fast K(+) channel (Kv1.1). DamID reveals that Sh is bound by both Isl and Lim3. Body wall muscle was used as a test tissue because in conditions of low Ca(2+), the fast K(+) current is carried solely by Sh channels (unlike neurons in which a second fast K(+) current, Shal, also contributes). Ectopic expression of isl, but not Lim3, is sufficient to reduce both Sh transcript and Sh current level. By contrast, coexpression of both TFs is additive, resulting in a significantly greater reduction in both Sh transcript and current compared with isl expression alone. These observations provide evidence for combinatorial activity of Isl and Lim3 in regulating ion channel gene expression.

Bhambhani, C., Ravindranath, A. J., Mentink, R. A., Chang, M. V., Betist, M. C., Yang, Y. X., Koushika, S. P., Korswagen, H. C. and Cadigan, K. M. (2014). Distinct DNA Binding Sites Contribute to the TCF Transcriptional Switch in C. elegans and Drosophila. PLoS Genet 10: e1004133. PubMed ID: 24516405
Summary: Regulation of gene expression by signaling pathways often occurs through a transcriptional switch, where the transcription factor responsible for signal-dependent gene activation represses the same targets in the absence of signaling. T-cell factors (TCFs) are transcription factors in the Wnt/β-catenin pathway, which control numerous cell fate specification events in metazoans. The TCF transcriptional switch is mediated by many co-regulators that contribute to repression or activation of Wnt target genes. It is typically assumed that DNA recognition by TCFs is important for target gene location, but plays no role in the actual switch. TCF/Pangolin (the fly TCF) and some vertebrate TCF isoforms bind DNA through two distinct domains, a High Mobility Group (HMG) domain and a C-clamp, which recognize DNA motifs known as HMG and Helper sites, respectively. This study demonstrates that POP-1 (the C. elegans TCF) also activates target genes through HMG and Helper site interactions. Helper sites enhanced the ability of a synthetic enhancer to detect Wnt/β-catenin signaling in several tissues and revealed an unsuspected role for POP-1 in regulating the C. elegans defecation cycle. Searching for HMG-Helper site clusters allowed the identification of a new POP-1 target gene active in the head muscles and gut. While Helper sites and the C-clamp are essential for activation of worm and fly Wnt targets, they are dispensable for TCF-dependent repression of targets in the absence of Wnt signaling. These data suggest that a fundamental change in TCF-DNA binding contributes to the transcriptional switch that occurs upon Wnt stimulation.

Drewell, R. A., Nevarez, M. J., Kurata, J. S., Winkler, L. N., Li, L. and Dresch, J. M. (2013). Deciphering the combinatorial architecture of a Drosophila homeotic gene enhancer. Mech Dev. PubMed ID: 24514265
Summary: In Drosophila, the 330 kb bithorax complex regulates cellular differentiation along the anterio-posterior axis during development in the thorax and abdomen and is comprised of three homeotic genes: Ultrabithorax, abdominal-A, and Abdominal-B. The expression of each of these genes is in turn controlled through interactions between transcription factors and a number of cis-regulatory modules in the neighboring intergenic regions. This study examine how the sequence architecture of transcription factor binding sites mediates the functional activity of one of these cis-regulatory modules. Using computational, mathematical modeling and experimental molecular genetic approaches the IAB7b enhancer, which regulates Abdominal-B expression specifically in the presumptive seventh and ninth abdominal segments of the early embryo, was investigated. A cross-species comparison of the IAB7b enhancer reveals an evolutionarily conserved signature motif containing two Fushi-Tarazu activator transcription factor binding sites. Yhe transcriptional repressors Knirps, Kruppel and Giant are able to restrict reporter gene expression to the posterior abdominal segments, using different molecular mechanisms including short-range repression and competitive binding. Additionally, the functional importance of the spacing between the two Fushi-Tarazu binding sites is shown, and the potential importance of cooperativity for transcriptional activation is discussed. These results demonstrate that the transcriptional output of the IAB7b cis-regulatory module relies on a complex set of combinatorial inputs mediated by specific transcription factor binding and that the sequence architecture at this enhancer is critical to maintain robust regulatory function.

Sunday, February 23rd

Nalabothula, N., McVicker, G., Maiorano, J., Martin, R., Pritchard, J. K. and Fondufe-Mittendorf, Y. N. (2014). The chromatin architectural proteins HMGD1 and H1 bind reciprocally and have opposite effects on chromatin structure and gene regulation. BMC Genomics 15: 92. PubMed ID: 24484546
Summary: Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. While these proteins are almost certainly important for gene regulation they have been studied far less than the core histone proteins. This study describes the genomic distributions and functional roles of two chromatin architectural proteins: histone H1 and the high mobility group protein HMGD1 in Drosophila S2 cells. Using ChIP-seq, biochemical and gene specific approaches, it was found that HMGD1 binds to highly accessible regulatory chromatin and active promoters. In contrast, H1 is primarily associated with heterochromatic regions marked with repressive histone marks. The ratio of HMGD1 to H1 binding is a better predictor of gene activity than either protein by itself, which suggests that reciprocal binding between these proteins is important for gene regulation. Using knockdown experiments, it was shown that HMGD1 and H1 affect the occupancy of the other protein, change nucleosome repeat length and modulate gene expression. Collectively, these data suggest that dynamic and mutually exclusive binding of H1 and HMGD1 to nucleosomes and their linker sequences may control the fluid chromatin structure that is required for transcriptional regulation. This study provides a framework to further study the interplay between chromatin architectural proteins and epigenetics in gene regulation.

Klinker, H., Mueller-Planitz, F., Yang, R., Forne, I., Liu, C. F., Nordenskiold, L. and Becker, P. B. (2014). ISWI Remodelling of Physiological Chromatin Fibres Acetylated at Lysine 16 of Histone H4. PLoS One 9: e88411. PubMed ID: 24516652
Summary: ISWI is the catalytic subunit of several ATP-dependent chromatin remodelling factors that catalyse the sliding of nucleosomes along DNA and thereby endow chromatin with structural flexibility. Full activity of ISWI requires residues of a basic patch of amino acids in the N-terminal 'tail' of histone H4. Previous studies employing oligopeptides and mononucleosomes suggested that acetylation of the H4 tail at lysine 16 (H4K16) within the basic patch may inhibit the activity of ISWI. On the other hand, the acetylation of H4K16 is known to decompact chromatin fibres. Conceivably, decompaction may enhance the accessibility of nucleosomal DNA and the H4 tail for ISWI interactions. Such an effect can only be evaluated at the level of nucleosome arrays. This study probed the influence of H4K16 acetylation on the ATPase and nucleosome sliding activity of Drosophila ISWI in the context of defined, in vitro reconstituted chromatin fibres with physiological nucleosome spacing and linker histone content. Contrary to widespread expectations, the acetylation did not inhibit ISWI activity, but rather stimulated ISWI remodelling under certain conditions. Therefore, the effect of H4K16 acetylation on ISWI remodelling depends on the precise nature of the substrate.

Liang, J., Lacroix, L., Gamot, A., Cuddapah, S., Queille, S., Lhoumaud, P., Lepetit, P., Martin, P. G., Vogelmann, J., Court, F., Hennion, M., Micas, G., Urbach, S., Bouchez, O., Nollmann, M., Zhao, K., Emberly, E. and Cuvier, O. (2014). Chromatin Immunoprecipitation Indirect Peaks Highlight Long-Range Interactions of Insulator Proteins and Pol II Pausing. Mol Cell. PubMed ID: 24486021
Summary: Eukaryotic chromosomes are partitioned into topologically associating domains (TADs) that are demarcated by distinct insulator-binding proteins (IBPs) in Drosophila. Whether IBPs regulate specific long-range contacts and how this may impact gene expression remains unclear. This study has identified 'indirect peaks' of multiple IBPs that represent their distant sites of interactions through long-range contacts. Indirect peaks depend on protein-protein interactions among multiple IBPs and their common cofactors, including CP190, as confirmed by high-resolution analyses of long-range contacts. Mutant IBPs unable to interact with CP190 impair long-range contacts as well as the expression of hundreds of distant genes that are specifically flanked by indirect peaks. Regulation of distant genes strongly correlates with RNAPII pausing, highlighting how this key transcriptional stage may trap insulator-based long-range interactions. These data illustrate how indirect peaks may decipher gene regulatory networks through specific long-range interactions.

Cuartero, S., Fresan, U., Reina, O., Planet, E. and Espinas, M. L. (2014). Ibf1 and Ibf2 are novel CP190-interacting proteins required for insulator function. EMBO J. PubMed ID: 24502977
Summary: Insulators are DNA-protein complexes that play a central role in chromatin organization and regulation of gene expression. In Drosophila different proteins, dCTCF, Su(Hw), and BEAF bind to specific subsets of insulators most of them having in common CP190. It has been shown that there are a number of CP190-binding sites that are not shared with any other known insulator protein, suggesting that other proteins could cooperate with CP190 to regulate insulator activity. This paper reports on the identification of two previously uncharacterized proteins as CP190-interacting proteins, that were named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190-binding sites throughout the genome. It was also show nthat Ibf1 and Ibf2 are DNA-binding proteins that form hetero-oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancer-blocking assays and Ibf2 null mutation cause a homeotic phenotype. Taken together these data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity.

Saturday, February 22nd

Johnston, R. J. and Desplan, C. (2014). Interchromosomal communication coordinates intrinsically stochastic expression between alleles. Science 343: 661-665. PubMed ID: 24503853
Summary: Sensory systems use stochastic mechanisms to diversify neuronal subtypes. In the Drosophila eye, stochastic expression of the PAS-bHLH transcription factor Spineless (Ss) determines a random binary subtype choice in R7 photoreceptors. This study shows that a stochastic, cell-autonomous decision to express ss is made intrinsically by each ss locus. Stochastic on or off expression of each ss allele is determined by combinatorial inputs from one enhancer and two silencers acting at long range. However, the two ss alleles also average their frequency of expression through up-regulatory and down-regulatory interallelic cross-talk. This inter- or intrachromosomal long-range regulation does not require endogenous ss chromosomal positioning or pairing. Therefore, although individual ss alleles make independent stochastic choices, interchromosomal communication coordinates expression state between alleles, ensuring that they are both expressed in the same random subset of R7s.

Wernet, M. F. and Desplan, C. (2014). Homothorax and Extradenticle alter the transcription factor network in Drosophila ommatidia at the dorsal rim of the retina. Development 141: 918-928. PubMed ID: 24496628
Summary: A narrow band of ommatidia in the dorsal periphery of the Drosophila retina called the dorsal rim area (DRA) act as detectors for polarized light. The transcription factor Homothorax (Hth) is expressed in DRA inner photoreceptors R7 and R8 and is both necessary and sufficient to induce the DRA fate, including specialized morphology and unique Rhodopsin expression. Hth expression is the result of Wingless (Wg) pathway activity at the eye margins and restriction to the dorsal eye by the selector genes of the Iroquois complex (Iro-C). However, how the DRA is limited to exactly one or two ommatidial rows is not known. Although several factors regulating the Drosophila retinal mosaic are expressed in DRA ommatidia, the role of Hth in this transcriptional network is uncharacterized. Here we show that Hth functions together with its co-factor Extradenticle (Exd) to repress the R8-specific factor Senseless (Sens) in DRA R8 cells, allowing expression of an ultraviolet-sensitive R7 Rhodopsin (Rh3). Furthermore, Hth/Exd act in concert with the transcriptional activators Orthodenticle (Otd) and Spalt (Sal), to activate expression of Rh3 in the DRA. The resulting monochromatic coupling of Rh3 between R7 and R8 in DRA ommatidia is important for comparing celestial e-vector orientation rather than wavelengths. Finally, Hth expression was shown to expand to many ommatidial rows in regulatory mutants of optomotor-blind (omb), a transcription factor transducing Wg signaling at the dorsal and ventral eye poles. Therefore, locally restricted recruitment of the DRA-specific factor Hth alters the transcriptional network that regulates Rhodopsin expression across ommatidia.

Li, C. R., Guo, D. and Pick, L. (2013). Independent signaling by Drosophila insulin receptor for axon guidance and growth. Front Physiol 4: 385. PubMed ID: 24478707
Summary: The Drosophila insulin receptor (DInR) regulates a diverse array of biological processes including growth, axon guidance, and sugar homeostasis. Growth regulation by DInR is mediated by Chico, the Drosophila homolog of vertebrate insulin receptor substrate proteins IRS1-4. In contrast, DInR regulation of photoreceptor axon guidance in the developing visual system is mediated by the SH2-SH3 domain adaptor protein Dreadlocks (Dock). In vitro studies by others identified five NPXY motifs, one in the juxtamembrane region and four in the signaling C-terminal tail (C-tail), important for interaction with Chico. This study used yeast two-hybrid assays to identify regions in the DInR C-tail that interact with Dock. These Dock binding sites were in separate portions of the C-tail from the previously identified Chico binding sites. To test whether these sites are required for growth or axon guidance in whole animals, a panel of DInR proteins, in which the putative Chico and Dock interaction sites had been mutated individually or in combination, were tested for their ability to rescue viability, growth and axon guidance defects of dinr mutant flies. Sites required for viability were identified. Unexpectedly, mutation of both putative Dock binding sites, either individually or in combination, did not lead to defects in photoreceptor axon guidance. Thus, either sites also required for viability are necessary for DInR function in axon guidance and/or there is redundancy built into the DInR/Dock interaction such that Dock is able to interact with multiple regions of DInR. It was also found that simultaneous mutation of all five NPXY motifs implicated in Chico interaction drastically decreased growth in both male and female adult flies. These animals resembled chico mutants, supporting the notion that DInR interacts directly with Chico in vivo to control body size. Mutation of these five NPXY motifs did not affect photoreceptor axon guidance, segregating the roles of DInR in the processes of growth and axon guidance.

Kang, J., Yeom, E., Lim, J. and Choi, K. W. (2014). Bar Represses dPax2 and Decapentaplegic to Regulate Cell Fate and Morphogenetic Cell Death in Drosophila Eye. PLoS One 9: e88171. PubMed ID: 24505414
Summary: The coordinated regulation of cell fate and cell survival is crucial for normal pattern formation in developing organisms. In Drosophila compound eye development, crystalline arrays of hexagonal ommatidia are established by precise assembly of diverse cell types, including the photoreceptor cells, cone cells and interommatidial (IOM) pigment cells. The molecular basis for controlling the number of cone and IOM pigment cells during ommatidial pattern formation is not well understood. This study presents evidence that BarH1 and BarH2 homeobox genes are essential for eye patterning by inhibiting excess cone cell differentiation and promoting programmed death of IOM cells. Specifically, loss of Bar from the undifferentiated retinal precursor cells was shown to lead to ectopic expression of Prospero and dPax2, two transcription factors essential for cone cell specification, resulting in excess cone cell differentiation. It was also shown that loss of Bar causes ectopic expression of the TGFbeta homolog Decapentaplegic (Dpp) posterior to the morphogenetic furrow in the larval eye imaginal disc. The ectopic Dpp expression is not responsible for the formation of excess cone cells in Bar loss-of-function mutant eyes. Instead, it causes reduction in IOM cell death in the pupal stage by antagonizing the function of pro-apoptotic gene reaper. Taken together, this study suggests a novel regulatory mechanism in the control of developmental cell death in which the repression of Dpp by Bar in larval eye disc is essential for IOM cell death in pupal retina.

Friday, February 21st

Karuppudurai, T., Lin, T. Y., Ting, C. Y., Pursley, R., Melnattur, K. V., Diao, F., White, B. H., Macpherson, L. J., Gallio, M., Pohida, T. and Lee, C. H. (2014). A Hard-Wired Glutamatergic Circuit Pools and Relays UV Signals to Mediate Spectral Preference in Drosophila. Neuron 81: 603-615. PubMed ID: 24507194
Summary: Many visual animals have innate preferences for particular wavelengths of light, which can be modified by learning. Drosophila's preference for UV over visible light requires UV-sensing R7 photoreceptors and specific wide-field amacrine neurons called Dm8. This study identified three types of medulla projection neurons downstream of R7 and Dm8 and showed that selectively inactivating one of them (Tm5c) abolishes UV preference. Using a modified GRASP method to probe synaptic connections at the single-cell level, it was revealed that each Dm8 neuron forms multiple synaptic contacts with Tm5c in the center of Dm8's dendritic field but sparse connections in the periphery. By single-cell transcript profiling and RNAi-mediated knockdown, it was determined that Tm5c uses the kainate receptor Clumsy to receive excitatory glutamate input from Dm8. It is concluded that R7s-->Dm8-->Tm5c form a hard-wired glutamatergic circuit that mediates UV preference by pooling approximately 16 R7 signals for transfer to the lobula, a higher visual center.

Meier, M., Serbe, E., Maisak, M. S., Haag, J., Dickson, B. J. and Borst, A. (2014). Neural Circuit Components of the Drosophila OFF Motion Vision Pathway. Curr Biol [Epub ahead of print]. PubMed ID: 24508173
Summary: Detecting the direction of visual motion is an essential task of the early visual system. The Reichardt detector has been proven to be a faithful description of the underlying computation in insects. A series of recent studies addressed the neural implementation of the Reichardt detector in Drosophila revealing the overall layout in parallel ON and OFF channels, its input neurons from the lamina (L1-->ON, and L2-->OFF), and the respective output neurons to the lobula plate (ON-->T4, and OFF-->T5). While anatomical studies showed that T4 cells receive input from L1 via Mi1 and Tm3 cells, the neurons connecting L2 to T5 cells have not been identified so far. It is, however, known that L2 contacts, among others, two neurons, called Tm2 and L4, which show a pronounced directionality in their wiring. This study characterized the visual response properties of both Tm2 and L4 neurons via Ca2+ imaging. It was found that Tm2 and L4 cells respond with an increase in activity to moving OFF edges in a direction-unselective manner. To investigate their participation in motion vision, their output was blocked while recording from downstream tangential cells in the lobula plate. Silencing of Tm2 and L4 completely abolishes the response to moving OFF edges. These results demonstrate that both cell types are essential components of the Drosophila OFF motion vision pathway, prior to the computation of directionality in the dendrites of T5 cells.

Wang, K., Gong, J., Wang, Q., Li, H., Cheng, Q., Liu, Y., Zeng, S. and Wang, Z. (2014). Parallel pathways convey olfactory information with opposite polarities in Drosophila. Proc Natl Acad Sci [Epub ahead of print]. PubMed ID: 24516124
Summary: In insects, olfactory information received by peripheral olfactory receptor neurons (ORNs) is conveyed from the antennal lobes (ALs) to higher brain regions by olfactory projection neurons (PNs). Despite the knowledge that multiple types of PNs exist, little is known about how these different neuronal pathways work cooperatively. This paper examined the Drosophila GABAergic mediolateral antennocerebral tract PNs (mlPNs), which link ipsilateral AL and lateral horn (LH), in comparison with the cholinergic medial tract PNs (mPNs). The connectivity of mlPNs in ALs was examined, and most mlPNs were found to receive inputs from both ORNs and mPNs and participate in AL network function by forming gap junctions with other AL neurons. Meanwhile, mlPNs might innervate LH neurons downstream of mPNs, exerting a feedforward inhibition. Using dual-color calcium imaging, which enables a simultaneous monitoring of neural activities in two groups of PNs, it was found that mlPNs exhibit robust odor responses overlapping with, but broader than, those of mPNs. Moreover, preferentially down-regulation of GABA in most mlPNs caused abnormal courtship and aggressive behaviors in male flies. These findings demonstrate that in Drosophila, olfactory information in opposite polarities are carried coordinately by two parallel and interacting pathways, which could be essential for appropriate behaviors.

Sakai, T., Watanabe, K., Ohashi, H., Sato, S., Inami, S., Shimada, N. and Kitamoto, T. (2014). Insulin-Producing Cells Regulate the Sexual Receptivity through the Painless TRP Channel in Drosophila Virgin Females. PLoS One 9: e88175. PubMed ID: 24505416
Summary: In a variety of animal species, females hold a leading position in evaluating potential mating partners. The decision of virgin females to accept or reject a courting male is one of the most critical steps for mating success. In the fruitfly Drosophila melanogaster, however, the molecular and neuronal mechanisms underlying female receptivity are still poorly understood, particularly for virgin females. The Drosophila painless (pain) gene encodes a transient receptor potential (TRP) ion channel. Previous studies have demonstrated that mutations in pain significantly enhance the sexual receptivity of virgin females and that pain expression in painGAL4-positive neurons is necessary and sufficient for pain-mediated regulation of the virgin receptivity. Among the painGAL4-positive neurons in the adult female brain, this study has found that insulin-producing cells (IPCs), a neuronal subset in the pars intercerebralis, are essential in virgin females for the regulation of sexual receptivity through Pain TRP channels. IPC-specific knockdown of pain expression or IPC ablation strongly enhanced female sexual receptivity as was observed in pain mutant females. When pain expression or neuronal activity was conditionally suppressed in adult IPCs, female sexual receptivity was similarly enhanced. Furthermore, both pain mutations and the conditional knockdown of pain expression in IPCs depressed female rejection behaviors toward courting males. Taken together, these results indicate that the Pain TRP channel in IPCs plays an important role in controlling the sexual receptivity of Drosophila virgin females by positively regulating female rejection behaviors during courtship.

Thursday, February 20th

Li, Z., Guo, Y., Han, L., Zhang, Y., Shi, L., Huang, X. and Lin, X. (2014). Debra-mediated ci degradation controls tissue homeostasis in Drosophila adult midgut. Stem Cell Reports 2: 135-144. PubMed ID: 24527387
Summary: Adult tissue homeostasis is maintained by resident stem cells and their progeny. However, the underlying mechanisms that control tissue homeostasis are not fully understood. The protein Debra has been shown to be involved in the Hedgehog signaling pathway as a mediator of protein degradation by the lysosome. This study demonstrates that Debra-mediated Ci degradation is important for intestinal stem cell (ISC) proliferation in Drosophila adult midgut. Debra inhibition leads to increased ISC activity and tissue homeostasis loss, phenocopying defects observed in aging flies. These defects can be suppressed by depleting Ci, suggesting that increased Hedgehog (Hh) signaling contributes to ISC proliferation and tissue homeostasis loss. Consistently, Hh signaling activation causes the same defects, whereas depletion of Hh signaling suppresses these defects. Furthermore, the Hh ligand from multiple sources is involved in ISC proliferation and tissue homeostasis. Finally, it was shown that the JNK pathway acts downstream of Hh signaling to regulate ISC proliferation. Together, these results provide insights into the mechanisms of stem cell proliferation and tissue homeostasis control.

Tsarouhas, V., Yao, L. and Samakovlis, C. (2014). Src-kinases and ERK activate distinct responses to Stitcher receptor tyrosine kinase signaling during wound healing in Drosophila. J Cell Sci [Epub ahead of print]. PubMed ID: 24522188
Summary: Metazoans have evolved efficient mechanisms for epidermal repair and survival upon injury. Several cellular responses and key signaling molecules involved in wound healing have been identified in Drosophila but the coordination of cytoskeletal rearrangements and the activation of gene expression during barrier repair is poorly understood. The Ret-like, receptor tyrosine kinase Stitcher (Stit; Cadherin 96Ca in FlyBase) regulates both re-epithelialization and transcriptional activation by Grainy head (Grh) to induce extracellular barrier restoration. This study describes the immediate down-stream effectors of Stit signaling in vivo. Drk (Downstream of receptor kinase) and Src-family tyrosine kinases (see Src oncogene at 42A) bind to the same docking site in the Stit intracellular domain. Drk is required for the full activation of transcriptional responses but is dispensable for re-epithelialization. By contrast, Src-family kinases control both the assembly of a contractile actin ring at the wound periphery and Grh-dependent activation of barrier repair genes. This analysis identifies distinct pathways mediating injury responses and reveals an RTK-dependent activation mode of Src-kinases and their central functions during epidermal wound healing in vivo.

Tulina, N. M., Chen, W. F., Chen, J. H., Sowcik, M. and Sehgal, A. (2014). Day-night cycles and the sleep-promoting factor, Sleepless, affect stem cell activity in the Drosophila testis. Proc Natl Acad Sci [Epub ahead of print]. PubMed ID: 24516136
Summary: Adult stem cells maintain tissue integrity and function by renewing cellular content of the organism through regulated mitotic divisions. Previous studies showed that stem cell activity is affected by local, systemic, and environmental cues. This study explored a role of environmental day-night cycles in modulating cell cycle progression in populations of stem cells of the adult testis. Using a classic stem cell system, the Drosophila spermatogonial stem cell niche, daily rhythms were revealed in division frequencies of germ-line and somatic stem cells that act cooperatively to produce male gametes. Whether behavioral sleep-wake cycles, which are driven by the environmental day-night cycles, regulate stem cell function, was also examined. Flies lacking the sleep-promoting factor Sleepless, which maintains normal sleep in Drosophila, were found to have increased germ-line stem cell (GSC) division rates, and this effect is mediated, in part, through a GABAergic signaling pathway. It is suggested that alterations in sleep can influence the daily dynamics of GSC divisions.

Baumbach, J., Hummel, P., Bickmeyer, I., Kowalczyk, K. M., Frank, M., Knorr, K., Hildebrandt, A., Riedel, D., Jackle, H. and Kuhnlein, R. P. (2014). A Drosophila in vivo screen identifies store-operated calcium entry as a key regulator of adiposity. Cell Metab 19: 331-343. PubMed ID: 24506874
Summary: To unravel the evolutionarily conserved genetic network underlying energy homeostasis, a systematic in vivo gene knockdown screen was performed in Drosophila. A transgenic RNAi library enriched for fly orthologs of human genes was used to functionally impair about half of all Drosophila genes specifically in adult fat storage tissue. This approach identified 77 genes that affect the body fat content of the fly, including 58 previously unknown obesity-associated genes. These genes function in diverse biological processes such as lipid metabolism, vesicle-mediated trafficking, and the universal store-operated calcium entry (SOCE). Impairment of the SOCE core component Stromal interaction molecule (Stim), as well as other components of the pathway, causes adiposity in flies. Acute Stim dysfunction in the fat storage tissue triggers hyperphagia via remote control of the orexigenic short neuropeptide F in the brain, which in turn affects the coordinated lipogenic and lipolytic gene regulation, resulting in adipose tissue hypertrophy.

Wednesday, February 19th

Okusawa, S., Kohsaka, H. and Nose, A. (2014). Serotonin and downstream leucokinin neurons modulate larval turning behavior in Drosophila. J Neurosci 34: 2544-2558. PubMed ID: 24523545
Summary: Serotonin (5-HT) is known to modulate motor outputs in a variety of animal behaviors. However, the downstream neural pathways of 5-HT remain poorly understood. This paper describes role of 5-HT in directional change, or turning, behavior of fruit fly larvae. Light- and touch-induced turning was analyzed and it was found that turning is a combination of three components: bending, retreating, and rearing. Serotonin transmission suppresses rearing; when 5-HT neurons were inhibited with Shibire or Kir2.1, rearing increased without affecting the occurrence of bending or retreating. Increased rearing in the absence of 5-HT transmission often results in slower or failed turning, indicating that suppression of rearing by 5-HT is critical for successful turning. A class of abdominal neurons, called the abdominal LK neurons (ABLKs), which express the 5-HT1B receptor and the neuropeptide leucokinin, was identified as downstream targets of 5-HT that are involved in the control of turning. Increased rearing was observed when neural transmission or leucokinin synthesis was inhibited in these cells. Forced activation of ABLKs also increased rearing, suggesting that an appropriate level of ABLK activity is critical for the control of turning. Calcium imaging revealed that ABLKs show periodic activation with an interval of approximately 15 s. The activity level of ABLKs increased and decreased in response to a 5-HT agonist and antagonist, respectively. These results suggest that 5-HT modulates larval turning by regulating the activity level of downstream ABLK neurons and secretion of the neuropeptide leucokinin.

van Breugel, F., Suver, M. P. and Dickinson, M. H. (2014). Octopaminergic modulation of the visual flight speed regulator of Drosophila. J Exp Biol [Epub ahead of print]. PubMed ID: 24526725
Summary: Recent evidence suggests that flies' sensitivity to large field optic flow is increased by the release of octopamine during flight. This increase in gain presumably enhances visually-mediated behaviors such as the active regulation of forward speed, a process that involves the comparison of a vision-based estimate of velocity with an internal set point. To determine where in the neural circuit this comparison is made, the octopamine neurons were selectively silenced in Drosophila and the effect on vision-based velocity regulation was examined in free flying flies. It was found that flies with inactivated octopamine neurons accelerate more slowly in response to visual motion than control flies, but maintained nearly the same baseline flight speed. These results are parsimonious with a circuit architecture in which the internal control signal is injected into the visual motion pathway upstream of the interneuron network that estimates groundspeed.

Ng, S. H., Shankar, S., Shikichi, Y., Akasaka, K., Mori, K. and Yew, J. Y. (2014). Pheromone evolution and sexual behavior in Drosophila are shaped by male sensory exploitation of other males. Proc Natl Acad Sci [Epub ahead of print]. PubMed ID: 24516141
Summary: Animals exhibit a spectacular array of traits to attract mates. Understanding the evolutionary origins of sexual features and preferences is a fundamental problem in evolutionary biology, and the mechanisms remain highly controversial. In some species, females choose mates based on direct benefits conferred by the male to the female and her offspring. Thus, female preferences are thought to originate and coevolve with male traits. In contrast, sensory exploitation occurs when expression of a male trait takes advantage of preexisting sensory biases in females. This study documents in Drosophila a previously unidentified example of sensory exploitation of males by other males through the use of the sex pheromone CH503 (see A new male sex-pheromone and novel cuticular cues for chemical communication in Drosophila). Mass spectrometry, high-performance liquid chromatography, and behavioral analysis were used to demonstrate that an antiaphrodisiac produced by males of the melanogaster subgroup also is effective in distant Drosophila relatives that do not express the pheromone. It was further shown that species that produce the pheromone have become less sensitive to the compound, illustrating that sensory adaptation occurs after sensory exploitation. These findings provide a mechanism for the origin of a sex pheromone and show that sensory exploitation changes male sexual behavior over evolutionary time.

Chung, H., Loehlin, D. W., Dufour, H. D., Vacarro, K., Millar, J. G. and Carroll, S. B. (2014). A Single Gene Affects Both Ecological Divergence and Mate Choice in Drosophila. Science [Epub ahead of print]. PubMed ID: 24526311
Summary: Evolutionary changes in traits involved in both ecological divergence and mate choice may produce reproductive isolation and speciation. However, there are few examples of such dual traits, and the genetic and molecular bases of their evolution have not been identified. This study shows that methyl-branched cuticular hydrocarbons (mbCHCs) are a dual trait that affects both desiccation resistance and mate choice in Drosophila serrata. A fatty acid synthase mFAS (CG3524) was identified that is responsible for mbCHC production in Drosophila; expression of mFAS is undetectable in oenocytes (cells that produce CHCs) of a closely related, desiccation-sensitive species, D. birchii, due in part to multiple changes in cis-regulatory sequences of mFAS. It is suggested that ecologically influenced changes in the production of mbCHCs have contributed to reproductive isolation between the two species.

Tuesday, February 18th

Zhu, J. Y., Vereshchagina, N., Sreekumar, V., Burbulla, L. F., Costa, A. C., Daub, K. J., Woitalla, D., Martins, L. M., Kruger, R. and Rasse, T. M. (2013). Knockdown of Hsc70-5/mortalin Induces Loss of Synaptic Mitochondria in a Drosophila Parkinson's Disease Model. PLoS One 8: e83714. PubMed ID: 24386261
Summary: Mortalin is an essential component of the molecular machinery that imports nuclear-encoded proteins into mitochondria, assists in their folding, and protects against damage upon accumulation of dysfunctional, unfolded proteins in aging mitochondria. Mortalin dysfunction associated with Parkinson's disease (PD) increases the vulnerability of cultured cells to proteolytic stress and leads to changes in mitochondrial function and morphology. To date, Drosophila melanogaster has been successfully used to investigate pathogenesis following the loss of several other PD-associated genes. This study generated the first loss-of-Hsc70-5/mortalin-function Drosophila model. The reduction of Mortalin expression recapitulates some of the defects observed in the existing Drosophila PD-models, which include reduced ATP levels, abnormal wing posture, shortened life span, and reduced spontaneous locomotor and climbing ability. Dopaminergic neurons seem to be more sensitive to the loss of mortalin than other neuronal sub-types and non-neuronal tissues. The loss of synaptic mitochondria is an early pathological change that might cause later degenerative events. It precedes both behavioral abnormalities and structural changes at the neuromuscular junction (NMJ) of mortalin-knockdown larvae that exhibit increased mitochondrial fragmentation. Autophagy is concomitantly up-regulated, suggesting that mitochondria are degraded via mitophagy. Ex vivo data from human fibroblasts identifies increased mitophagy as an early pathological change that precedes apoptosis. Given the specificity of the observed defects, it can be confidently said that the loss-of-mortalin model presented in this study will be useful for further dissection of the complex network of pathways that underlie the development of mitochondrial parkinsonism.

Abe, M., Naqvi, A., Hendriks, G. J., Feltzin, V., Zhu, Y., Grigoriev, A. and Bonini, N. M. (2014). Impact of age-associated increase in 2'-O-methylation of miRNAs on aging and neurodegeneration in Drosophila. Genes Dev 28: 44-57. PubMed ID: 24395246
Summary: MicroRNAs (miRNAs) are 20- to approximately 24-nucleotide (nt) small RNAs that impact a variety of biological processes, from development to age-associated events. To study the role of miRNAs in aging, studies have profiled the levels of miRNAs with time. However, evidence suggests that miRNAs show heterogeneity in length and sequence in different biological contexts. By examining the expression pattern of miRNAs by Northern blot analysis, this study found that Drosophila miRNAs show distinct isoform pattern changes with age. Surprisingly, an increase of some miRNAs reflects increased 2'-O-methylation of select isoforms. Small RNA deep sequencing revealed a global increase of miRNAs loaded into Ago2, but not into Ago1, with age. The data suggest increased loading of miRNAs into Ago2, but not Ago1, with age, indicating a mechanism for differential loading of miRNAs with age between Ago1 and Ago2. Mutations in Hen1 and Ago2, which lack 2'-O-methylation of miRNAs, result in accelerated neurodegeneration and shorter life span, suggesting a potential impact of the age-associated increase of 2'-O-methylation of small RNAs on age-associated processes. This study highlights that miRNA 2'-O-methylation at the 3' end is modulated by differential partitioning of miRNAs between Ago1 and Ago2 with age and that this process, along with other functions of Ago2, might impact age-associated events in Drosophila.

Tufi, R., et al. (2014). Enhancing nucleotide metabolism protects against mitochondrial dysfunction and neurodegeneration in a PINK1 model of Parkinson's disease. Nat Cell Biol [Epub ahead of print]. PubMed ID: 24441527
Summary: Mutations in PINK1 cause early-onset Parkinson's disease (PD). Studies in Drosophila melanogaster have highlighted mitochondrial dysfunction on loss of Pink1 as a central mechanism of PD pathogenesis. This study shows that global analysis of transcriptional changes in Drosophila pink1 mutants reveals an upregulation of genes involved in nucleotide metabolism, critical for neuronal mitochondrial DNA synthesis. These key transcriptional changes were also detected in brains of PD patients harbouring PINK1 mutations. Genetic enhancement of the nucleotide salvage pathway in neurons of pink1 mutant flies rescues mitochondrial impairment. In addition, pharmacological approaches enhancing nucleotide pools reduce mitochondrial dysfunction caused by Pink1 deficiency. It is concluded that loss of Pink1 evokes the activation of a previously unidentified metabolic reprogramming pathway to increase nucleotide pools and promote mitochondrial biogenesis. It is proposed that targeting strategies enhancing nucleotide synthesis pathways may reverse mitochondrial dysfunction and rescue neurodegeneration in PD and, potentially, other diseases linked to mitochondrial impairment.

Klein, P., Muller-Rischart, A. K., Motori, E., Schonbauer, C., Schnorrer, F., Winklhofer, K. F. and Klein, R. (2014). Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants. EMBO J. PubMed ID: 24473149
Summary: Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. This study investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. It was shown that signaling active version of Ret (RetMEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of RetMEN2B significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD.

Monday, February 17th

Bulat, V., Rast, M. and Pielage, J. (2014). Presynaptic CK2 promotes synapse organization and stability by targeting Ankyrin2. J Cell Biol 204: 77-94. PubMed ID: 24395637:
The precise regulation of synapse maintenance is critical to the development and function of neuronal circuits. Using an in vivo RNAi screen targeting the Drosophila kinome and phosphatome, eleven kinases and phosphatases were identified controlling synapse stability by regulating cytoskeletal, phospholipid, or metabolic signaling. This study focused on casein kinase 2 (CK2) and demonstrates that the regulatory (β) and catalytic (α) subunits of CK2 are essential for synapse maintenance. CK2α kinase activity is required in the presynaptic motoneuron, and its interaction with CK2β mediates cooperatively by two N-terminal residues of CK2α and is essential for CK2 holoenzyme complex stability and function in vivo. Using genetic and biochemical approaches Ankyrin2 was identified as a key presynaptic target of CK2 to maintain synapse stability. In addition, CK2 activity controls the subcellular organization of individual synaptic release sites within the presynaptic nerve terminal. This study identifies phosphorylation of structural synaptic components as a compelling mechanism to actively control the development and longevity of synaptic connections.

Kittelmann, M., Hegermann, J., Goncharov, A., Taru, H., Ellisman, M. H., Richmond, J. E., Jin, Y. and Eimer, S. (2013. Liprin-alpha/SYD-2 determines the size of dense projections in presynaptic active zones in C. elegans. J Cell Biol 203: 849-863. PubMed ID: 24322429
Summary: Synaptic vesicle (SV) release is spatially and temporally regulated by a network of proteins that form the presynaptic active zone (AZ). The hallmark of most AZs is an electron-dense projection (DP) surrounded by SVs. Despite their importance for understanding of triggered SV release, high-resolution analyses of DP structures are limited. Using electron microscopy, this study shows that DPs at Caenorhabditis elegans neuromuscular junctions (NMJs) were highly structured, composed of building units forming bays in which SVs are docked to the AZ membrane. Furthermore, larger ribbonlike DPs that were multimers of the NMJ building unit are found at synapses between inter- and motoneurons. It was also demonstrated that DP size is determined by the activity of the AZ protein SYD-2/Liprin-alpha (see Drosophila Liprin-α). Whereas loss of syd-2 function led to smaller DPs, syd-2 gain-of-function mutants displayed larger ribbonlike DPs through increased recruitment of ELKS-1/ELKS. Therefore, these data suggest that a main role of SYD-2/Liprin-alpha in synaptogenesis is to regulate the polymerization of DPs.

Morel, V., Lepicard, S., A, N. R., Parmentier, M. L. and Schaeffer, L. (2014). Drosophila Nesprin-1 controls glutamate receptor density at neuromuscular junctions. Cell Mol Life Sci. [Epub ahead of print]. PubMed ID: 24492984
Summary: Nesprin-1 is a core component of a protein complex connecting nuclei to cytoskeleton termed LINC (linker of nucleoskeleton and cytoskeleton). Nesprin-1 is anchored to the nuclear envelope by its C-terminal KASH domain, the disruption of which has been associated with neuronal and neuromuscular pathologies, including autosomal recessive cerebellar ataxia and Emery-Dreifuss muscular dystrophy. This study describes a new and unexpected role of Drosophila Nesprin-1, Msp-300, in neuromuscular junction. Larvae carrying a deletion of Msp-300 KASH domain (Msp-300ΔKASH) present a locomotion defect suggestive of a myasthenia, and demonstrate the importance of muscle Msp-300 for this phenotype, using tissue-specific RNAi knock-down. Msp-300ΔKASH mutants display abnormal neurotransmission at the larval neuromuscular junction, as well as an imbalance in postsynaptic glutamate receptor composition with a decreased percentage of GluRIIA-containing receptors. Msp-300ΔKASH locomotion phenotypes could be rescued by GluRIIA overexpression, suggesting that the locomotion impairment associated with the KASH domain deletion is due to a reduction in junctional GluRIIA. In summary, this study found that Msp-300 controls GluRIIA density at the neuromuscular junction. Theses results suggest that Drosophila is a valuable model for further deciphering how Nesprin-1 and LINC disruption may lead to neuronal and neuromuscular pathologies.

Mahoney, R. E., Rawson, J. M. and Eaton, B. A. (2014). An age-dependent change in the set point of synaptic homeostasis. J Neurosci 34: 2111-2119. PubMed ID: 24501352
Summary: Homeostatic plasticity functions within the nervous system to maintain normal neural functions, such as neurotransmission, within predefined optimal ranges. The defined output of these neuronal processes is referred to as the set point, which is the value that the homeostatic system defends against fluctuations. Currently, it is unknown how stable homeostatic set points are within the nervous system. The present study used the CM9 neuromuscular junctions (NMJs) in the adult Drosophila to investigate the stability of the set point of synaptic homeostasis across the lifespan of the fly. At the fly NMJ, it is believed that the depolarization of the muscle by neurotransmitter during an action potential, represented by the EPSP, is a homeostatic set point that is precisely maintained via changes in synaptic vesicle release. This study found that the amplitude of the EPSP abruptly increases during middle age and that this enhanced EPSP is maintained into late life, consistent with an age-dependent change to the homeostatic set point of the synapse during middle age. In support of this, comparison of the homeostatic response at the young versus the old synapse shows that the magnitude of the homeostatic response at the older synapse is significantly larger than the response at the young NMJ, appropriate for a synapse at which the set point has been increased. These data demonstrate that the amplitude of the EPSP at the Drosophila NMJ increases during aging and that the homeostatic signaling system adjusts its response to accommodate the new set point.

Sunday, February 16th

Lee, K. S., Wu, Z., Song, Y., Mitra, S. S., Feroze, A. H., Cheshier, S. H. and Lu, B. (2013). Roles of PINK1, mTORC2, and mitochondria in preserving brain tumor-forming stem cells in a noncanonical Notch signaling pathway. Genes Dev 27: 2642-2647. PubMed ID: 24352421
Summary: The self-renewal versus differentiation choice of Drosophila and mammalian neural stem cells (NSCs) requires Notch (N) signaling. How N regulates NSC behavior is not well understood. This study shows that canonical N signaling cooperates with a noncanonical N signaling pathway to mediate N-directed NSC regulation. In the noncanonical pathway, N interacts with PTEN-induced kinase 1 (PINK1) to influence mitochondrial function, activating mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling (see the TOR signaling pathway). Importantly, attenuating noncanonical N signaling preferentially impairs the maintenance of Drosophila and human cancer stem cell-like tumor-forming cells. These results emphasize the importance of mitochondria to N and NSC biology, with important implications for diseases associated with aberrant N signaling

Hakim, Y., Yaniv, S. P. and Schuldiner, O. (2014). Astrocytes play a key role in Drosophila mushroom body axon pruning. PLoS One 9: e86178. PubMed ID: 24465945
Summary: Axon pruning is an evolutionarily conserved strategy used to remodel neuronal connections during development. The Drosophila mushroom body (MB) undergoes neuronal remodeling in a highly stereotypical and tightly regulated manner, however many open questions remain. Although it has been previously shown that glia instruct pruning by secreting a TGF-beta ligand, myoglianin, which primes MB neurons for fragmentation and also later engulf the axonal debris once fragmentation has been completed, which glia subtypes participate in these processes as well as the molecular details are unknown. This study shows that, unexpectedly, astrocytes are the major glial subtype that is responsible for the clearance of MB axon debris following fragmentation, even though they represent only a minority of glia in the MB area during remodeling. Furthermore, astrocytes both promote fragmentation of MB axons as well as clear axonal debris and that this process is mediated by ecdysone signaling in the astrocytes themselves. In addition, this study found that blocking the expression of the cell engulfment receptor Draper in astrocytes affects only axonal debris clearance. Thereby this study uncoupled the function of astrocytes in promoting axon fragmentation to that of clearing axonal debris after fragmentation has been completed. This study finds a novel role for astrocytes in the MB and suggests two separate pathways in which they affect developmental axon pruning.

Ting, C. Y., McQueen, P. G., Pandya, N., Lin, T. Y., Yang, M., Reddy, O. V., O'Connor, M. B., McAuliffe, M. and Lee, C. H. (2014). Photoreceptor-Derived Activin Promotes Dendritic Termination and Restricts the Receptive Fields of First-Order Interneurons in Drosophila. Neuron [Epub ahead of print]. PubMed ID: 24462039
Summary: How neurons form appropriately sized dendritic fields to encounter their presynaptic partners is poorly understood. The Drosophila medulla is organized in layers and columns and innervated by medulla neuron dendrites and photoreceptor axons. This study shows that three types of medulla projection (Tm) neurons extend their dendrites in stereotyped directions and to distinct layers within a single column for processing retinotopic information. In contrast, the Dm8 amacrine neurons form a wide dendritic field to receive approximately 16 R7 photoreceptor inputs. R7- and R8-derived Activin selectively restricts the dendritic fields of their respective postsynaptic partners, Dm8 and Tm20, to the size appropriate for their functions. Canonical Activin signaling promotes dendritic termination without affecting dendritic routing direction or layer. Tm20 neurons lacking Activin signaling expande their dendritic fields and aberrantly synapse with neighboring photoreceptors. It is suggested that afferent-derived Activin regulates the dendritic field size of their postsynaptic partners to ensure appropriate synaptic partnership.

King, I. F., Eddison, M., Kaun, K. R. and Heberlein, U. (2014). EGFR and FGFR Pathways Have Distinct Roles in Drosophila Mushroom Body Development and Ethanol-Induced Behavior. PLoS One 9: e87714. PubMed ID: 24498174
Summary: Epidermal Growth Factor Receptor (EGFR) signaling has a conserved role in ethanol-induced behavior in flies and mice, affecting ethanol-induced sedation in both species. However it is not known what other effects EGFR signaling may have on ethanol-induced behavior, or what roles other Receptor Tyrosine Kinase (RTK) pathways may play in ethanol induced behaviors. This study examined the effects of both the EGFR and Fibroblast Growth Factor Receptor (FGFR) RTK signaling pathways on ethanol-induced enhancement of locomotion, a behavior distinct from sedation that may be associated with the rewarding effects of ethanol. Both EGFR and FGFR genes were found to influence ethanol-induced locomotion, though their effects are opposite - EGFR signaling suppresses this behavior, while FGFR signaling promotes it. EGFR signaling affects development of the Drosophila mushroom bodies in conjunction with the JNK MAP kinase basket (bsk), and with the Ste20 kinase Tao, and it is hypothesized that the EGFR pathway affects ethanol-induced locomotion through its effects on neuronal development. It was found, however, that FGFR signaling most likely affects ethanol-induced behavior through a different mechanism, possibly through acute action in adult neurons.

Saturday, February 15th

Ravindranath, A. and Cadigan, K. M. (2014). Structure-Function Analysis of the C-clamp of TCF/Pangolin in Wnt/β-catenin Signaling. PLoS One 9: e86180. PubMed ID: 24465946
Summary: The evolutionarily conserved Wnt/β-catenin pathway plays an important role in animal development in metazoans. Many Wnt targets are regulated by members of the TCF/LEF1 (TCF) family of transcription factors. All TCFs contain a High Mobility Group (HMG) domain that bind specific DNA sequences. Invertebrate TCFs and some vertebrate TCF isoforms also contain another domain, called the C-clamp, which allows TCFs to recognize an additional DNA motif known as the Helper site. While the C-clamp has been shown to be important for regulating several Wnt reporter genes in cell culture, its physiological role in regulating Wnt targets is less clear. In addition, little is known about this domain, except that two of the four conserved cysteines are functionally important. This study carried out a systematic mutagenesis and functional analysis of the C-clamp from the Drosophila TCF/Pangolin (TCF/Pan) protein. The C-clamp was found to be a zinc-binding domain that is sufficient for binding to the Helper site. In addition to this DNA-binding activity, the C-clamp also inhibits the HMG domain from binding its cognate DNA site. Point mutations were identified that specifically affected DNA-binding or reduced the inhibitory effect. These mutants were characterized in TCF/Pan rescue assays. The specific DNA-binding activity of the C-clamp was shown to be essential for TCF/Pan function in cell culture and in patterning the embryonic epidermis of Drosophila, demonstrating the importance of this C-clamp activity in regulating Wnt target gene expression. In contrast, the inhibitory mutation had a subtle effect in cell culture and no effect on TCF/Pan activity in embryos. These results provide important information about the functional domains of the C-clamp, and highlight its importance for Wnt/β-cat signaling in Drosophila.

Okenve-Ramos, P. and Llimargas, M. (2014). Fascin links Btl/FGFR signalling to the actin cytoskeleton during Drosophila tracheal morphogenesis. Development 141: 929-939. PubMed ID: 24496629
Summary: A key challenge in normal development and in disease is to elucidate the mechanisms of cell migration. This question was approached using the tracheal system of Drosophila as a model. Tracheal cell migration requires the Breathless/FGFR pathway; however, how the pathway induces migration remains poorly understood. The Breathless pathway was found to upregulate singed at the tip of tracheal branches, and this regulation was found to be functionally relevant. singed encodes Drosophila Fascin, which belongs to a conserved family of actin-bundling proteins involved in cancer progression and metastasis upon misregulation. singed was shown to be required for filopodia stiffness and proper morphology of tracheal tip cells, defects that correlate with an abnormal actin organisation. It is proposed that singed-regulated filopodia and cell fronts are required for timely and guided branch migration and for terminal branching and branch fusion. singed requirements were shown to rely on its actin-bundling activity controlled by phosphorylation, and that active Singed can promote tip cell features. Furthermore, singed was found to act in concert with forked, another actin cross-linker. The absence of both cross-linkers further stresses the relevance of tip cell morphology and filopodia for tracheal development. In summary, these results on the one hand reveal a previously undescribed role for forked in the organisation of transient actin structures such as filopodia, and on the other hand identify singed as a new target of Breathless signal, establishing a link between guidance cues, the actin cytoskeleton and tracheal morphogenesis.

Hall, S., Bone, C., Oshima, K., Zhang, L., McGraw, M., Lucas, B., Fehon, R. G. and Ward, R. E. t. (2014). Macroglobulin complement-related encodes a protein required for septate junction organization and paracellular barrier function in Drosophila. Development 141: 889-898. PubMed ID: 24496625
Summary: Polarized epithelia play crucial roles as barriers to the outside environment and enable the formation of specialized compartments for organs to carry out essential functions. Barrier functions are mediated by cellular junctions that line the lateral plasma membrane between cells, principally tight junctions in vertebrates and septate junctions (SJs) in invertebrates. Over the last two decades, more than 20 genes have been identified that function in SJ biogenesis in Drosophila, including those that encode core structural components of the junction such as Neurexin IV, Coracle and several claudins, as well as proteins that facilitate the trafficking of SJ proteins during their assembly. This study demonstrates that Macroglobulin complement-related (Mcr), a gene previously implicated in innate immunity, plays an essential role during embryonic development in SJ organization and function. Mcr was shown to colocalize with other SJ proteins in mature ectodermally derived epithelial cells, that it shows interdependence with other SJ proteins for SJ localization, and that Mcr mutant epithelia fail to form an effective paracellular barrier. Tissue-specific RNA interference further demonstrates that Mcr is required cell-autonomously for SJ organization. Finally, a unique interdependence between Mcr and Neuroglian was shown for SJ localization that provides new insights into the organization of the SJ. Together, these studies demonstrate that Mcr is a core component of epithelial SJs and also highlight an interesting relationship between innate immunity and epithelial barrier functions.

Batz, T., Forster, D. and Luschnig, S. (2014). The transmembrane protein Macroglobulin complement-related is essential for septate junction formation and epithelial barrier function in Drosophila. Development 141: 899-908. PubMed ID: 24496626
Summary: Occluding cell-cell junctions in epithelia form physical barriers that separate different membrane domains, restrict paracellular diffusion and prevent pathogens from spreading across tissues. In invertebrates, these functions are provided by septate junctions (SJs), the functional equivalent of vertebrate tight junctions. How the diverse functions of SJs are integrated and modulated in a multiprotein complex is not clear, and many SJ components are still unknown. This study reports the identification of Macroglobulin complement-related (Mcr), a member of the conserved α-2-macroglobulin complement protein family, as a novel SJ-associated protein in Drosophila. Whereas α-2-macroglobulin complement proteins are generally known as secreted factors that bind to surfaces of pathogens and target them for phagocytic uptake, Mcr represents an unusual α-2-macroglobulin protein with a predicted transmembrane domain. Mcr protein was shown to localize to lateral membranes of epithelial cells, where its distribution overlaps with SJs. Several SJ components are required for the correct localization of Mcr. Conversely, Mcr is required in a cell-autonomous fashion for the correct membrane localization of SJ components, indicating that membrane-bound rather than secreted Mcr isoforms are involved in SJ formation. Finally, loss of Mcr function was shown to lead to morphological, ultrastructural and epithelial barrier defects resembling mutants lacking SJ components. These results, along with previous findings on the role of Mcr in phagocytosis, suggest that Mcr plays dual roles in epithelial barrier formation and innate immunity. Thus, Mcr represents a novel paradigm for investigating functional links between occluding junction formation and pathogen defense mechanisms.

Friday, February 14th

Shahbazi, M. N., Megias, D., Epifano, C., Akhmanova, A., Gundersen, G. G., Fuchs, E. and Perez-Moreno, M. (2013). CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions. J Cell Biol 203: 1043-1061. PubMed ID: 24368809
Summary: Classical cadherins and their connections with microtubules (MTs) are emerging as important determinants of cell adhesion. However, the functional relevance of such interactions and the molecular players that contribute to tissue architecture are still emerging. This paper reports that the MT plus end-binding protein CLASP2 localizes to adherens junctions (AJs) via direct interaction with p120-catenin [p120; (see Drosophila p120ctn)] in primary basal mouse keratinocytes. Reductions in the levels of p120 or CLASP2 decreased the localization of the other protein to cell-cell contacts and altered AJ dynamics and stability. These features were accompanied by decreased MT density and altered MT dynamics at intercellular junction sites. Interestingly, CLASP2 was enriched at the cortex of basal progenitor keratinocytes, in close localization to p120. These findings suggest the existence of a new mechanism of MT targeting to AJs with potential functional implications in the maintenance of proper cell-cell adhesion in epidermal stem cells.

Franz, A., Roque, H., Saurya, S., Dobbelaere, J. and Raff, J. W. (2013). CP110 exhibits novel regulatory activities during centriole assembly in Drosophila. J Cell Biol 203: 785-799. PubMed ID: 24297749
Summary: CP110 is a conserved centriole protein implicated in the regulation of cell division, centriole duplication, and centriole length and in the suppression of ciliogenesis. Surprisingly, mutant flies lacking CP110 (CP110Δ) are viable and fertile and have no obvious defects in cell division, centriole duplication, or cilia formation. This study shows that CP110 has at least three functions in flies. First, it subtly influences centriole length by counteracting the centriole-elongating activity of several centriole duplication proteins. Specifically, centrioles are ~10% longer than normal in CP110Δ mutants and ~20% shorter when CP110 is overexpressed. Second, CP110 ensures that the centriolar microtubules do not extend beyond the distal end of the centriole, as some centriolar microtubules can be more than 50 times longer than the centriole in the absence of CP110. Finally, and unexpectedly, CP110 suppresses centriole overduplication induced by the overexpression of centriole duplication proteins. These studies identify novel and surprising functions for CP110 in vivo in flies.

Kobayashi, T., Kim, S., Lin, Y. C., Inoue, T. and Dynlacht, B. D. (2014). The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly. J Cell Biol 204: 215-229. PubMed ID: 24421332
Summary: Talpid3/KIAA0586 has been identified as a component of a CP110-containing protein complex important for centrosome and cilia function. Talpid3 assembles a ring-like structure at the extreme distal end of centrioles. Ablation of Talpid3 results in an aberrant distribution of centriolar satellites involved in protein trafficking to centrosomes as well as cilia assembly defects, reminiscent of loss of Cep290, another CP110-associated protein. Talpid3 depletion also leads to mislocalization of Rab8a, a small GTPase thought to be essential for ciliary vesicle formation. Expression of activated Rab8a suppresses cilia assembly defects provoked by Talpid3 depletion, suggesting that Talpid3 affects cilia formation through Rab8a recruitment and/or activation. Remarkably, ultrastructural analyses showed that Talpid3 is required for centriolar satellite dispersal, which precedes the formation of mature ciliary vesicles, a process requiring Cep290. These studies suggest that Talpid3 and Cep290 play overlapping and distinct roles in ciliary vesicle formation through regulation of centriolar satellite accretion and Rab8a.

Lesniewska, K., Warbrick, E. and Ohkura, H. (2014). Peptide aptamers define distinct EB1- and EB3-binding motifs and interfere with microtubule dynamics. Mol Biol Cell [Epub ahead of print]. PubMed ID: 24478452
Summary: EB1 is a conserved protein that plays a central role in regulating microtubule dynamics and organisation. It binds directly to microtubule plus ends and recruits other plus end localising proteins. Most EB1-binding proteins contain an SxIP (Ser-any residue-Ile-Pro) motif. This study describes the isolation of peptide aptamers with optimised versions of this motif by screening for interaction with the Drosophila EB1 protein. The use of small peptide aptamers to competitively inhibit protein interaction and function is becoming increasingly recognised as a powerful technique. SxIP aptamers were shown to bind microtubule plus ends in cells and can functionally act to displace interacting proteins by competitive binding. Their expression in developing flies can interfere with microtubules, altering their dynamics. Aptamers were identified that bind to human EB1 and EB3, which revealed sequence requirements similar to but distinct from each other and from Drosophila EB1. This suggests that EB1 paralogues within one species may interact with overlapping but distinct sets of proteins in cells.

Thursday, February 13th

Chen, C. C., Dechassa, M. L., Bettini, E., Ledoux, M. B., Belisario, C., Heun, P., Luger, K. and Mellone, B. G. (2014). CAL1 is the Drosophila CENP-A assembly factor. J Cell Biol. PubMed ID: 24469636
Summary: Centromeres are specified epigenetically by the incorporation of the histone H3 variant CENP-A. In humans, amphibians, and fungi, CENP-A is deposited at centromeres by the HJURP/Scm3 family of assembly factors, but homologues of these chaperones are absent from a number of major eukaryotic lineages such as insects, fish, nematodes, and plants. In Drosophila, centromeric deposition of CENP-A requires the fly-specific protein CAL1. This study shows that targeting CAL1 to noncentromeric DNA in Drosophila cells is sufficient to heritably recruit CENP-A, kinetochore proteins, and microtubule attachments. CAL1 selectively interacts with CENP-A and is sufficient to assemble CENP-A nucleosomes that display properties consistent with left-handed octamers. The CENP-A assembly activity of CAL1 resides within an N-terminal domain, whereas the C terminus mediates centromere recognition through an interaction with CENP-C. Collectively, this work identifies the 'missing' CENP-A chaperone in flies, revealing fundamental conservation between insect and vertebrate centromere-specification mechanisms.

Coelho, P. A., Bury, L., Sharif, B., Riparbelli, M. G., Fu, J., Callaini, G., Glover, D. M. and Zernicka-Goetz, M. (2013). Spindle formation in the mouse embryo requires plk4 in the absence of centrioles. Dev Cell 27: 586-597. PubMed ID: 24268700
Summary: During the first five rounds of cell division in the mouse embryo, spindles assemble in the absence of centrioles. Spindle formation initiates around chromosomes, but the microtubule nucleating process remains unclear. This study demonstrates that Plk4 (see Drosophila SAK), a protein kinase known as a master regulator of centriole formation, is also essential for spindle assembly in the absence of centrioles. Depletion of maternal Plk4 prevents nucleation and growth of microtubules and results in monopolar spindle formation. This leads to cytokinesis failure and, consequently, developmental arrest. Plk4 function depends on its kinase activity and its partner protein, Cep152 (see Drosophila Asterless). Moreover, tethering Cep152 to cellular membranes sequesters Plk4 and is sufficient to trigger spindle assembly from ectopic membranous sites. Thus, the Plk4-Cep152 complex has an unexpected role in promoting microtubule nucleation in the vicinity of chromosomes to mediate bipolar spindle formation in the absence of centrioles.

Schweizer, N., Ferras, C., Kern, D. M., Logarinho, E., Cheeseman, I. M. and Maiato, H. (2013). Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis. J Cell Biol. PubMed ID: 24344181
Summary: Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. This study shows that Tpr (see Drosophila Megator) is required for normal SAC response by stabilizing Mad1 (see Drosophila Mad1) and Mad2 (see Drosophila Mad2) before mitosis. Tpr coimmunoprecipitates with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1-c-Mad2 recruitment to NPCs. Interestingly, Tpr is normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1-c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restores normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, evidence is provided that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response.

Cuylen, S., Metz, J., Hruby, A. and Haering, C. H. (2013). Entrapment of chromosomes by condensin rings prevents their breakage during cytokinesis. Dev Cell 27: 469-478. PubMed ID: 24286828
Summary: Successful segregation of chromosomes during mitosis and meiosis depends on the action of the ring-shaped condensin complex (see Barren), but how condensin ensures the complete disjunction of sister chromatids is unknown. This study shows that the failure to segregate chromosome arms, which results from condensin release from chromosomes by proteolytic cleavage of its ring structure, leads to a DNA damage checkpoint-dependent cell-cycle arrest. Checkpoint activation is triggered by the formation of chromosome breaks during cytokinesis, which proceeds with normal timing despite the presence of lagging chromosome arms. Remarkably, enforcing condensin ring reclosure by chemically induced dimerization just before entry into anaphase is sufficient to restore chromosome arm segregation. It is suggested that topological entrapment of chromosome arms by condensin rings ensures their clearance from the cleavage plane and thereby avoids their breakage during cytokinesis.

Wednesday, February 12th

Ahanger, S. H., Gunther, K., Weth, O., Bartkuhn, M., Bhonde, R. R., Shouche, Y. S. and Renkawitz, R. (2014). Ectopically tethered CP190 induces large-scale chromatin decondensation. Sci Rep 4: 3917. PubMed ID: 24472778
Summary: Insulator mediated alteration in higher-order chromatin and/or nucleosome organization is an important aspect of epigenetic gene regulation. Recent studies have suggested a key role for CP190 in such processes. This study analysed the effects of ectopically tethered insulator factors on chromatin structure and found that CP190 induces large-scale decondensation when targeted to a condensed lacO array in mammalian and Drosophila cells. In contrast, dCTCF alone, is unable to cause such a decondensation, however, when CP190 is present, dCTCF recruits it to the lacO array and mediates chromatin unfolding. The CP190 induced opening of chromatin may not be correlated with transcriptional activation, as binding of CP190 does not enhance luciferase activity in reporter assays. It is proposed that CP190 may mediate histone modification and chromatin remodelling activity to induce an open chromatin state by its direct recruitment or targeting by a DNA binding factor such as dCTCF.

Neuman, S. D., Ihry, R. J., Gruetzmacher, K. M. and Bashirullah, A. (2014). INO80-dependent regression of ecdysone-induced transcriptional responses regulates developmental timing in Drosophila. Dev Biol. [Epub ahead of print]. PubMed ID: 24468295
Summary: Sequential pulses of the steroid hormone ecdysone regulate the major developmental transitions in Drosophila, and the duration of each developmental stage is determined by the length of time between ecdysone pulses. Ecdysone regulates biological responses by directly initiating target gene transcription. In turn, these transcriptional responses are known to be self-limiting, with mechanisms in place to ensure regression of hormone-dependent transcription. However, the biological significance of these transcriptional repression mechanisms remains unclear. This study shows that the chromatin remodeling protein Ino80 facilitates transcriptional repression of ecdysone-regulated genes during prepupal development. In ino80 mutant animals, inefficient repression of transcriptional responses to the late larval ecdysone pulse delays the onset of the subsequent prepupal ecdysone pulse, resulting in a significantly longer prepupal stage. Conversely, increased expression of ino80 is sufficient to shorten the prepupal stage by increasing the rate of transcriptional repression. Furthermore, it was demonstrated that enhancing the rate of regression of the mid-prepupal competence factor βFTZ-F1 is sufficient to determine the timing of head eversion and thus the duration of prepupal development. Although Ino80 is conserved from yeast to humans, this study represents the first characterization of a bona fide ino80 mutation in any metazoan, raising the possibility that the functions of Ino80 in transcriptional repression and developmental timing are evolutionarily conserved.

Wang, S. H., Nan, R., Accardo, M. C., Sentmanat, M., Dimitri, P. and Elgin, S. C. (2014). A Distinct Type of Heterochromatin at the Telomeric Region of the Drosophila melanogaster Y Chromosome. PLoS One 9: e86451. PubMed ID: 24475122
Summary: Heterochromatin assembly and its associated phenotype, position effect variegation (PEV), provide an informative system to study chromatin structure and genome packaging. In the fruit fly Drosophila melanogaster, the Y chromosome is entirely heterochromatic in all cell types except the male germline; as such, Y chromosome dosage is a potent modifier of PEV. However, neither Y heterochromatin composition, nor its assembly, has been carefully studied. This study reports the mapping and characterization of eight reporter lines that show male-specific PEV. In all eight cases, the reporter insertion sites lie in the telomeric transposon array (HeT-A and TART-B2 homologous repeats) of the Y chromosome short arm (Ys). Investigations of the impact on the PEV phenotype of mutations in known heterochromatin proteins (i.e., modifiers of PEV) show that this Ys telomeric region is a unique heterochromatin domain: it displays sensitivity to mutations in HP1a, Eggless and SU(VAR)3-9, but no sensitivity to Su(z)2 mutations. It appears that the endo-siRNA pathway plays a major targeting role for this domain. Interestingly, an ectopic copy of 1360 is sufficient to induce a piRNA targeting mechanism to further enhance silencing of a reporter cytologically localized to the Ys telomere. These results demonstrate the diversity of heterochromatin domains, and the corresponding variation in potential targeting mechanisms.

Mohan, R. D., Dialynas, G., Weake, V. M., Liu, J., Martin-Brown, S., Florens, L., Washburn, M. P., Workman, J. L. and Abmayr, S. M. (2014). Loss of Drosophila Ataxin-7, a SAGA subunit, reduces H2B ubiquitination and leads to neural and retinal degeneration. Genes Dev 28: 259-272. PubMed ID: 24493646
Summary: The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex possesses acetyltransferase and deubiquitinase activities. Within this modular complex, Ataxin-7 anchors the deubiquitinase activity to the larger complex. This study identified and characterized Drosophila Ataxin-7 (CG9866) and found that reduction of Ataxin-7 protein results in loss of components from the SAGA complex. In contrast to yeast, where loss of Ataxin-7 inactivates the deubiquitinase and results in increased H2B ubiquitination, loss of Ataxin-7 results in decreased H2B ubiquitination and H3K9 acetylation without affecting other histone marks. Interestingly, the effect on ubiquitination was conserved in human cells, suggesting a novel mechanism regulating histone deubiquitination in higher organisms. Consistent with this mechanism in vivo, this study found that a recombinant deubiquitinase module is active in the absence of Ataxin-7 in vitro. When the consequences of reduced Ataxin-7 were examined in vivo, it was found that flies exhibited pronounced neural and retinal degeneration, impaired movement, and early lethality.

Tuesday, February 11th

Sha, K., Choi, S. H., Im, J., Lee, G. G., Loeffler, F. and Park, J. H. (2014). Regulation of Ethanol-Related Behavior and Ethanol Metabolism by the Corazonin Neurons and Corazonin Receptor in Drosophila melanogaster. PLoS One 9: e87062. PubMed ID: 24489834
Summary: Impaired ethanol metabolism can lead to various alcohol-related health problems. Key enzymes in ethanol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH); however, neuroendocrine pathways that regulate the activities of these enzymes are largely unexplored. This study has identified a neuroendocrine system involving Corazonin (Crz) neuropeptide and its receptor (CrzR) as important physiological regulators of ethanol metabolism in Drosophila. Crz-cell deficient (Crz-CD) flies displayed significantly delayed recovery from ethanol-induced sedation that is referred to as hangover-like phenotype. Newly generated mutant lacking Crz Receptor (CrzR01) and CrzR-knockdown flies showed even more severe hangover-like phenotype, which is causally associated with fast accumulation of acetaldehyde in theCrzR01 mutant following ethanol exposure. Higher levels of acetaldehyde are likely due to 30% reduced ALDH activity in the mutants. Moreover, increased ADH activity was found in the CrzR01 mutant, but not in the Crz-CD flies. Quantitative RT-PCR revealed transcriptional upregulation of Adh gene in the CrzR01. Transgenic inhibition of cyclic AMP-dependent protein kinase (PKA) also results in significantly increased ADH activity and Adh mRNA levels, indicating PKA-dependent transcriptional regulation of Adh by CrzR. Furthermore, inhibition of PKA or cAMP response element binding protein (CREB) in CrzR cells leads to comparable hangover-like phenotype to the CrzR01 mutant. These findings suggest that CrzR-associated signaling pathway is critical for ethanol detoxification via Crz-dependent regulation of ALDH activity and Crz-independent transcriptional regulation of ADH. This study provides new insights into the neuroendocrine-associated ethanol-related behavior and metabolism.

Simoni, A., Wolfgang, W., Topping, M. P., Kavlie, R. G., Stanewsky, R. and Albert, J. T. (2014). A mechanosensory pathway to the Drosophila circadian clock. Science 343: 525-528. PubMed ID: 24482478
Summary: Circadian clocks attune the physiology of virtually all living organisms to the diurnal cycles of their environments. In metazoan animals, multiple sensory input pathways have been linked to clock synchronization with the environmental cycle (entrainment). Extrinsic entrainment cues include light and temperature. This study shows that (12-hour:12-hour) cycles of vibration and silence (VS) are sufficient to synchronize the daily locomotor activity of wild-type Drosophila melanogaster. Behavioral synchronization to VS cycles required a functional clock and functional chordotonal organs and was accompanied by phase-shifts of the daily oscillations of Period protein concentrations in brain clock neurons. The feedback from mechanosensory-and particularly, proprioceptive-organs may help an animal to keep its circadian clock in sync with its own, stimulus-induced activities.

Apostolopoulou, A. A., Mazija, L., Wust, A. and Thum, A. S. (2014). The neuronal and molecular basis of quinine-dependent bitter taste signaling in Drosophila larvae. Front Behav Neurosci 8: 6. PubMed ID: 24478653
Summary: The sensation of bitter substances can alert an animal that a specific type of food is harmful and should not be consumed. However, not all bitter compounds are equally toxic and some may even be beneficial in certain contexts. Thus, taste systems in general may have a broader range of functions than just in alerting the animal. This study investigated bitter sensing and processing in Drosophila larvae using quinine, a substance perceived by humans as bitter. Behavioral choice, feeding, survival, and associative olfactory learning are all directly affected by quinine. On the cellular level, 12 gustatory sensory receptor neurons that express both GR66a and GR33a were shown to be required for quinine-dependent choice and feeding behavior. Interestingly, these neurons are not necessary for quinine-dependent survival or associative learning. On the molecular receptor gene level, the GR33a receptor, but not GR66a, is required for quinine-dependent choice behavior. A screen for gustatory sensory receptor neurons that trigger quinine-dependent choice behavior revealed that a single GR97a receptor gene expressing neuron located in the peripheral terminal sense organ is partially necessary and sufficient. For the first time, this study shows that the elementary chemosensory system of the Drosophila larva can serve as a simple model to understand the neuronal basis of taste information processing on the single cell level with respect to different behavioral outputs.

Barth, J., Dipt, S., Pech, U., Hermann, M., Riemensperger, T. and Fiala, A. (2014). Differential Associative Training Enhances Olfactory Acuity in Drosophila melanogaster. J Neurosci 34: 1819-1837. PubMed ID: 24478363
Summary: Training can improve the ability to discriminate between similar, confusable stimuli, including odors. One possibility of enhancing behaviorally expressed discrimination (i.e., sensory acuity) relies on differential associative learning, during which animals are forced to detect the differences between similar stimuli. Drosophila represents a key model organism for analyzing neuronal mechanisms underlying both odor processing and olfactory learning. However, the ability of flies to enhance fine discrimination between similar odors through differential associative learning has not been analyzed in detail. This study performed associative conditioning experiments using chemically similar odorants that were shown to evoke overlapping neuronal activity in the fly's antennal lobes and highly correlated activity in mushroom body lobes. The animals' performance was compared in discriminating between these odors after subjecting them to one of two types of training: either absolute conditioning, in which only one odor is reinforced, or differential conditioning, in which one odor is reinforced and a second odor is explicitly not reinforced. First, it was shown that differential conditioning decreases behavioral generalization of similar odorants in a choice situation. Second, it was demonstrated that this learned enhancement in olfactory acuity relies on both conditioned excitation and conditioned inhibition. Third, inhibitory local interneurons in the antennal lobes are shown to be required for behavioral fine discrimination between the two similar odors. Fourth, differential, but not absolute, training causes decorrelation of odor representations in the mushroom body. In conclusion, differential training with similar odors ultimately induces a behaviorally expressed contrast enhancement between the two similar stimuli that facilitates fine discrimination.

Monday, February 10th

Goergen, P., Kasagiannis, A., Schioth, H. B. and Williams, M. J. (2014). The Drosophila Small GTPase Rac2 is Required for Normal Feeding and Mating Behaviour. Behav Genet. [Epub ahead of print] PubMed ID: 24488496
Summary: All multicellular organisms require the ability to regulate bodily processes in order to maintain a stable condition, which necessitates fluctuations in internal metabolics, as well as modifications of outward behaviour. Understanding the genetics behind this modulation is important as a general model for the metabolic modification of behaviour. This study demonstrates that the activity of the small GTPase Rac2 is required in Drosophila for the proper regulation of lipid storage and feeding behaviour, as well as aggression and mating behaviours. Rac2 mutant males and females are susceptible to starvation and contain considerably less lipids than controls. Furthermore, Rac2 mutants also have disrupted feeding behaviour, eating fewer but larger meals than controls. Intriguingly, Rac2 mutant males rarely initiate aggressive behaviour and display significantly increased levels of courtship behaviour towards other males and mated females. From these results it is concluded that Rac2 has a central role in regulating the Drosophila homeostatic system.

Bjordal, M., Arquier, N., Kniazeff, J., Pin, J. P. and Leopold, P. (2014). Sensing of amino acids in a dopaminergic circuitry promotes rejection of an incomplete diet in Drosophila. Cell 156: 510-521. PubMed ID: 24485457
Summary: The brain is the central organizer of food intake, matching the quality and quantity of the food sources with organismal needs. To ensure appropriate amino acid balance, many species reject a diet lacking one or several essential amino acids (EAAs) and seek out a better food source. This study shows that, in Drosophila larvae, this behavior relies on innate sensing of amino acids in dopaminergic (DA) neurons of the brain. The amino acid sensor GCN2/eIF2α Kinase acts upstream of GABA signaling in DA neurons to promote avoidance of the EAA-deficient diet. Using real-time calcium imaging in larval brains, this study shows that amino acid imbalance induces a rapid and reversible activation of three DA neurons that are necessary and sufficient for food rejection. Taken together, these data identify a central amino-acid-sensing mechanism operating in specific DA neurons and controlling food intake.

Shi, M., Yue, Z., Kuryatov, A., Lindstrom, J. M. and Sehgal, A. (2014). Identification of Redeye, a new sleep-regulating protein whose expression is modulated by sleep amount. Elife 3: e01473. PubMed ID: 24497543
Summary: This study reports a new protein involved in the homeostatic regulation of sleep in Drosophila. A forward genetic screen was conducted of chemically mutagenized flies to identify short-sleeping mutants, and one, redeye (rye; nicotinic Acetylcholine Receptor α4), was found that shows a severe reduction of sleep length. Cloning of rye reveals that it encodes a nicotinic acetylcholine receptor alpha subunit required for Drosophila sleep. Levels of RYE oscillate in light-dark cycles and peak at times of daily sleep. Cycling of RYE is independent of a functional circadian clock, but rather depends upon the sleep homeostat, as protein levels are up-regulated in short-sleeping mutants and also in wild type animals following sleep deprivation. It is proposed that the homeostatic drive to sleep increases levels of RYE, which responds to this drive by promoting sleep.

Mauss, A. S., Meier, M., Serbe, E. and Borst, A. (2014). Optogenetic and pharmacologic dissection of feedforward inhibition in Drosophila motion vision. J Neurosci 34: 2254-2263. PubMed ID: 24501364
Summary: Visual systems extract directional motion information from spatiotemporal luminance changes on the retina. An algorithmic model, the Reichardt detector, accounts for this by multiplying adjacent inputs after asymmetric temporal filtering. The outputs of two mirror-symmetrical units tuned to opposite directions are thought to be subtracted on the dendrites of wide-field motion-sensitive lobula plate tangential cells by antagonistic transmitter systems. In Drosophila, small-field T4/T5 cells in the optic lobe carry visual motion information to the tangential cells that are depolarized during preferred and hyperpolarized during null direction motion. While preferred direction input is likely provided by excitation from T4/T5 terminals, the origin of null direction inhibition is unclear. Probing the connectivity between T4/T5 and tangential cells in Drosophila using a combination of optogenetics, electrophysiology, and pharmacology, this study found a direct excitatory as well as an indirect inhibitory component. This suggests that the null direction response is caused by feedforward inhibition via yet unidentified neurons.

Sunday, February 9th

Asahina, K., Watanabe, K., Duistermars, B. J., Hoopfer, E., Gonzalez, C. R., Eyjolfsdottir, E. A., Perona, P. and Anderson, D. J. (2014). Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila. Cell 156: 221-235. PubMed ID: 24439378
Summary: Males of most species are more aggressive than females, but the neural mechanisms underlying this dimorphism are not clear. This study identified a neuron and a gene that control the higher level of aggression characteristic of Drosophila melanogaster males. Males, but not females, contain a small cluster of FruM+ neurons that express the neuropeptide Tachykinin (Tk). Activation and silencing of these neurons increased and decreased, respectively, intermale aggression without affecting male-female courtship behavior. Mutations in both Tk and a candidate receptor, Takr86C, suppress the effect of neuronal activation, whereas overexpression of Tk potentiates it. Tk neuron activation overcomes reduced aggressiveness caused by eliminating a variety of sensory or contextual cues, suggesting that it promotes aggressive arousal or motivation. Tachykinin/Substance P has been implicated in aggression in mammals, including humans. Thus, the higher aggressiveness of Drosophila males reflects the sexually dimorphic expression of a neuropeptide that controls agonistic behaviors across phylogeny.

Lu, B., Zelle, K. M., Seltzer, R., Hefetz, A. and Ben-Shahar, Y. (2014). Feminization of pheromone-sensing neurons affects mating decisions in Drosophila males. Biol Open. [Epub ahead of print]. PubMed ID: 24463366
Summary: The response of individual animals to mating signals depends on the sexual identity of the individual and the genetics of the mating targets, which represent the mating social context (social environment). However, how social signals are sensed and integrated during mating decisions remains a mystery. One of the models for understanding mating behaviors in molecular and cellular terms is the male courtship ritual in the fruit fly (Drosophila melanogaster). A subset of gustatory receptor neurons (GRNs) that are enriched in the male appendages and express the ion channel ppk23 play a major role in the initiation and maintenance of male courtship via the perception of cuticular contact pheromones, and are likely to represent the main chemosensory pathway that influences mating decisions by males. This study shows that genetic feminization of ppk23-expressing GRNs in male flies resulted in a significant increase in male-male sexual attraction without an apparent impact on sexual attraction to females. Furthermore, it was shown that this increase in male-male sexual attraction is sensory specific, which can be modulated by variable social contexts. Finally, feminization of ppk23-expressing sensory neurons was shown to lead to major transcriptional shifts, which may explain the altered interpretation of the social environment by feminized males. Together, these data indicate that the sexual cellular identity of pheromone sensing GRNs plays a major role in how individual flies interpret their social environment in the context of mating decisions.

Ignatious Raja, J. S., Katanayeva, N., Katanaev, V. L. and Galizia, C. G. (2014). Role of G subgroup of G proteins in olfactory signaling of Drosophila melanogaster. Eur J Neurosci. [Epub ahead of print] PubMed ID: 24443946
Summary: Intracellular signaling in insect olfactory receptor neurons remains unclear, with both metabotropic and ionotropic components being discussed. This study investigated the role of heterotrimeric Go and Gi proteins using a combined behavioral, in vivo and in vitro approach. Specifically, it was shown that inhibiting Go in sensory neurons by pertussis toxin leads to behavioral deficits. The olfactory receptor dOr22a was heterologously expressed in human embryonic kidney cells (HEK293T). Stimulation with an odor led to calcium influx, which was amplified via calcium release from intracellular stores. Subsequent experiments indicated that the signaling was mediated by the Gβγ subunits of the heterotrimeric Go/i proteins. Finally, using in vivo calcium imaging, it was shown that Go and Gi contribute to odor responses both for the fast (phasic) as for the slow (tonic) response component. A transduction cascade model involving several parallel processes is proposed, in which the metabotropic component is activated by Go and Gi, and uses Gβγ.

van Swinderen, B. and Kottler, B. (2014). Explaining general anesthesia: A two-step hypothesis linking sleep circuits and the synaptic release machinery. Bioessays [Epub ahead of print]. PubMed ID: 24449137
Summary: Several general anesthetics produce their sedative effect by activating endogenous sleep pathways. It is proposed that general anesthesia is a two-step process targeting sleep circuits at low doses, and synaptic release mechanisms across the entire brain at the higher doses required for surgery. This hypothesis synthesizes data from a variety of model systems, some which require sleep (e.g. rodents and adult flies) and others that probably do not sleep (e.g. adult nematodes and cultured cell lines). Non-sleeping systems can be made insensitive (or hypersensitive) to some anesthetics by modifying a single pre-synaptic protein, syntaxin1A. This suggests that the synaptic release machinery, centered on the highly conserved SNARE complex, is an important target of general anesthetics in all animals. A careful consideration of SNARE architecture uncovers a potential mechanism for general anesthesia, which may be the primary target in animals that do not sleep, but a secondary target in animals that sleep.

Saturday, February 8th

Pagano, J. M., Kwak, H., Waters, C. T., Sprouse, R. O., White, B. S., Ozer, A., Szeto, K., Shalloway, D., Craighead, H. G. and Lis, J. T. (2014). Defining NELF-E RNA Binding in HIV-1 and Promoter-Proximal Pause Regions. PLoS Genet 10: e1004090. PubMed ID: 24453987
Summary: The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Pol II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. This study used in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. A non-conserved region within the RRM was also identified that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, promoter-proximal regions were analyzed genome-wide in Drosophila, and it was shown that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, a significant increase in NBEs was observed among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.

Ninova, M., Ronshaugen, M. and Griffiths-Jones, S. (2014). Fast-evolving microRNAs are highly expressed in the early embryo of Drosophila virilis. RNA [Epub ahead of print], PubMed ID: 24448446
Summary: MicroRNAs are short non-protein-coding RNAs that regulate gene expression at the post-transcriptional level and are essential for the embryonic development of multicellular animals. Comparative genome-scale analyses have revealed that metazoan evolution is accompanied by the continuous acquisition of novel microRNA genes. This suggests that novel microRNAs may promote innovation and diversity in development. The evolutionary origins were determined of extant Drosophila microRNAs, and the sequence divergence between the 130 orthologous microRNAs was estimated in Drosophila melanogaster and Drosophila virilis, separated by 63 million years of evolution. Small RNA sequencing data sets covering D. virilis development were generated, and the relationship was explored between microRNA conservation and expression in a developmental context. Late embryonic, larval, and adult stages were found to be dominated by conserved microRNAs. This pattern, however, does not hold for the early embryo, where rapidly evolving microRNAs are uniquely present at high levels in both species. The group of fast-evolving microRNAs that are highly expressed in the early embryo belong to two Drosophilid lineage-specific clusters: mir-310-313 and mir-309 approximately 6. These clusters have particularly complex evolutionary histories of duplication, gain, and loss. These analyses suggest that the early embryo is a more permissive environment for microRNA changes and innovations. Fast-evolving microRNAs, therefore, have the opportunity to become preferentially integrated in early developmental processes, and may impact the evolution of development. The relationship between microRNA conservation and expression throughout the development of Drosophila differs from that previously observed for protein-coding genes.

Yue, Y., Li, G., Yang, Y., Zhang, W., Pan, H., Chen, R., Shi, F. and Jin, Y. (2013). Regulation of Dscam exon 17 alternative splicing by steric hindrance in combination with RNA secondary structures. RNA Biol 10: 1822-1833. PubMed ID: 24448213
Summary: The gene Down syndrome cell adhesion molecule (Dscam) potentially encodes 38,016 distinct isoforms in Drosophila melanogaster via mutually exclusive splicing. This study revealed a combinatorial mechanism of regulation of Dscam exon 17 mutually exclusive splicing through steric hindrance in combination with RNA secondary structure. This mutually exclusive behavior is enforced by steric hindrance, due to the close proximity of the exon 17.2 branch point to exon 17.1 in Diptera, and the interval size constraint in non-Dipteran species. Moreover, intron-exon RNA structures are evolutionarily conserved in 36 non-Drosophila species of six distantly related orders (Diptera, Lepidoptera, Coleoptera, Hymenoptera, Hemiptera, and Phthiraptera), which regulates the selection of exon 17 variants via masking the splice site. By contrast, a previously uncharacterized RNA structure specifically activates exon 17.1 by bringing splice sites closer together in Drosophila, while the other moderately suppresses exon 17.1 selection by hindering the accessibility of polypyrimidine sequences. Taken together, these data suggest a phylogeny of increased complexity in regulating alternative splicing of Dscam exon 17 spanning more than 300 million years of insect evolution. These results also provide models of the regulation of alternative splicing through steric hindrance in combination with dynamic structural codes.

Luhur, A., Chawla, G., Wu, Y. C., Li, J. and Sokol, N. S. (2014). Drosha-independent DGCR8/Pasha pathway regulates neuronal morphogenesis. Proc Natl Acad Sci U S A 111: 14226. PubMed ID: 24474768
Summary: Cleavage of microRNAs and mRNAs by Drosha and its cofactor Pasha/DGCR8 is required for animal development, but whether these proteins also have independent roles in development has been unclear. Known phenotypes associated with loss of either one of these two proteins are very similar and consistent with their joint function, even though both cofactors are involved with additional distinct RNA biogenesis pathways. This study reports clear phenotypic differences between drosha and pasha/dgcr8 null alleles in two postembryonic lineages in the Drosophila brain: elimination of pasha/dgcr8 leads to defects that are not shared by drosha null mutations in the morphology of γ neurons in the mushroom body lineage, as well as many neurons in the anterodorsal projection neuron lineage. These morphological defects are not detected in neurons that are genetically depleted of two additional microRNA pathway components, dicer-1 and argonaute1, indicating that they are not due to loss of microRNA activity. They are, however, phenocopied by a newly identified recessive gain-of-function allele in drosha that probably interferes with the microRNA independent functions of Pasha/DGCR8. These data therefore identify a general Drosha-independent DGCR8/Pasha pathway that promotes proper morphology in multiple neuronal lineages. Given that reduction of human DGCR8/Pasha may contribute to the cognitive and behavioral characteristics of DiGeorge syndrome patients, disruption of this newly described pathway could underlie human neurological disease.

Friday, February 7th

Huang, H., Li, J., Hu, L., Ge, L., Ji, H., Zhao, Y. and Zhang, L. (2014). Bantam is essential for Drosophila intestinal stem cell proliferation in response to Hippo signaling. Dev Biol 385: 211-219. PubMed ID: 24262985
Summary: The Drosophila midgut has emerged as an attractive model system to study stem cell biology. Extensive studies have been carried out to investigate the mechanisms of how the signaling pathways integrate to regulate intestinal stem cells (ISCs), yet, whether the microRNAs are involved in ISC self-renewal and maintenance is unknown. This study demonstrates that the bantam microRNA is expressed specifically at high levels in Drosophila midgut precursor cells (including ISCs and enteroblasts) and secretory enteroendocrine cells while at extremely low levels in enterocytes. Furthermore, overexpression of bantam microRNA results in increase of the division of the midgut precursor cells, whereas loss of bantam microRNA decreases their proliferation. The mechanical studies show that bantam microRNA is essential for the Hpo pathway induced cell-autonomous ISC self-renewal, while it is disposable for EGFR and Notch pathways mediated ISC proliferation. More interestingly, bantam microRNA was found to not be required for the Hpo pathway mediated non-cell-autonomous ISC proliferation, revealing a novel mechanism by which the Hpo signaling pathway specifies its transcriptional targets in specific tissue to exhibit its biological functions.

Chen, L., Dumelie, J. G., Li, X., Cheng, M. H., Yang, Z., Laver, J. D., Siddiqui, N. U., Westwood, J. T., Morris, Q., Lipshitz, H. D. and Smibert, C. A. (2014). Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein. Genome Biol 15: R4. PubMed ID: 24393533
Summary: Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. To gain a panoramic view of Smaug function in the early embryo, mRNAs were identified that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. mRNAs were also identified that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug's target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.

Zhang, Q., Shalaby, N. A. and Buszczak, M. (2014). Changes in rRNA transcription influence proliferation and cell fate within a stem cell lineage. Science 343: 298-301. PubMed ID: 24436420
Summary: Ribosome biogenesis drives cell growth and proliferation, but mechanisms that modulate this process within specific lineages remain poorly understood. This study identified a Drosophila RNA polymerase I (Pol I) regulatory complex composed of Under-developed (Udd; CG18316), RNA polymerase I subunit B [TAF1B (CG6241)], and a TAF1C-like factor (CG10496). Disruption of udd or TAF1B results in reduced ovarian germline stem cell (GSC) proliferation. Female GSCs display high levels of ribosomal RNA (rRNA) transcription, and Udd becomes enriched in GSCs relative to their differentiating daughters. Increasing Pol I transcription delays differentiation, whereas reducing rRNA production induces both morphological changes that accompany multicellular cyst formation and specific decreased expression of the bone morphogenetic protein (BMP) pathway component Mad. These findings demonstrate that modulating rRNA synthesis fosters changes in the cell fate, growth, and proliferation of female Drosophila GSCs and their daughters.

Mirkovic-Hosle, M. and Forstemann, K. (2014). Transposon Defense by Endo-siRNAs, piRNAs and Somatic pilRNAs in Drosophila: Contributions of Loqs-PD and R2D2. PLoS One 9: e84994. PubMed ID: 24454776
Summary: Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing activities that target transposons: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves the Dicer-2 co-factors Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2, which primarily helps to direct siRNAs into the RNA interference effector Ago2. Nonetheless, loss of either protein is not sufficient to produce a phenotype comparable with a dcr-2 mutation. This study provides further deep sequencing evidence supporting the notion that R2D2 and Loqs-PD have partially overlapping function. Certain transposons display a preference for either dsRBD-protein during production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, this study confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs) that show a ping-pong RNA amplification cycle signature. Expression of the Piwi-family protein mRNAs are detected only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway.

Thursday, February 6th

Neville, M. C., et al. (2014). Male-Specific Fruitless Isoforms Target Neurodevelopmental Genes to Specify a Sexually Dimorphic Nervous System. Curr Biol. PubMed ID: 24440396
Summary: In Drosophila, male courtship behavior is regulated in large part by the gene fruitless (fru). fru encodes a set of putative transcription factors that promote male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. Little is known about how Fru proteins function at the level of transcriptional regulation or the role that isoform diversity plays in the formation of a male-specific nervous system. To characterize the roles of sex-specific Fru isoforms in specifying male behavior, this study generated novel isoform-specific mutants and used a genomic approach to identify direct Fru isoform targets during development. All Fru isoforms were shown to directly target genes involved in the development of the nervous system, with individual isoforms exhibiting unique binding specificities. fru behavioral phenotypes are specified by either a single isoform or a combination of isoforms. Finally, the utility of these data for the identification of novel sexually dimorphic genomic enhancers and novel downstream regulators of male sexual behavior is illustrated in this study. These findings suggest that Fru isoform diversity facilitates both redundancy and specificity in gene expression, and that the regulation of neuronal developmental genes may be the most ancient and conserved role of fru in the specification of a male-specific nervous system.

Linneweber, G. A., et al. (2014). Neuronal Control of Metabolism through Nutrient-Dependent Modulation of Tracheal Branching. Cell 156: 69-83. PubMed ID: 24439370
Summary: During adaptive angiogenesis, a key process in the etiology and treatment of cancer and obesity, the vasculature changes to meet the metabolic needs of its target tissues. Although the cues governing vascular remodeling are not fully understood, target-derived signals are generally believed to underlie this process. This study identified an alternative mechanism by characterizing the previously unrecognized nutrient-dependent plasticity of the Drosophila tracheal system: a network of oxygen-delivering tubules developmentally akin to mammalian blood vessels. This plasticity, particularly prominent in the intestine, drives-rather than responds to-metabolic change. Mechanistically, it is regulated by distinct populations of nutrient- and oxygen-responsive neurons that, through delivery of both local and systemic insulin- and VIP-like neuropeptides (see Insulin-related peptide and Pdf), sculpt the growth of specific tracheal subsets. Thus, a novel mechanism is described by which nutritional cues modulate neuronal activity to give rise to organ-specific, long-lasting changes in vascular architecture.

Ferreira, T., Ou, Y., Li, S., Giniger, E. and van Meyel, D. J. (2014). Dendrite architecture organized by transcriptional control of the F-actin nucleator Spire. Development 141: 650-660. PubMed ID: 24449841
Summary: The architectures of dendritic trees are crucial for the wiring and function of neuronal circuits because they determine coverage of receptive territories, as well as the nature and strength of sensory or synaptic inputs. This study describes a cell-intrinsic pathway sculpting dendritic arborization (da) neurons in Drosophila that requires Longitudinals Lacking (Lola), a BTB/POZ transcription factor, and its control of the F-actin cytoskeleton through Spire (Spir), an actin nucleation protein. Loss of Lola from da neurons reduced the overall length of dendritic arbors, increased the expression of Spir, and produced inappropriate F-actin-rich dendrites at positions too near the cell soma. Selective removal of Lola from only class IV da neurons decreased the evasive responses of larvae to nociception. The increased Spir expression contributed to the abnormal F-actin-rich dendrites and the decreased nocifensive responses because both were suppressed by reduced dose of Spir. Thus, an important role of Lola is to limit expression of Spir to appropriate levels within da neurons. Spir was found to be expressed in dendritic arbors and to be important for their development. Removal of Spir from class IV da neurons reduced F-actin levels and total branch number, shifted the position of greatest branch density away from the cell soma, and compromised nocifensive behavior. It is concluded that the Lola-Spir pathway is crucial for the spatial arrangement of branches within dendritic trees and for neural circuit function because it provides balanced control of the F-actin cytoskeleton.

Freeman, E. G., Wisotsky, Z. and Dahanukar, A. (2014). Detection of sweet tastants by a conserved group of insect gustatory receptors. Proc Natl Acad Sci U S A 111: 1598-1603. PubMed ID: 24474785
Summary: Sweet taste cells play critical roles in food selection and feeding behaviors. Drosophila sweet neurons express eight gustatory receptors (Grs) belonging to a highly conserved clade in insects. Despite ongoing efforts, little is known about the fundamental principles that underlie how sweet tastants are detected by these receptors. This study provides systematic functional analysis of Drosophila sweet receptors using the ab1C CO2-sensing olfactory neuron as a unique in vivo decoder. Each of the eight receptors of this group was found to confer sensitivity to one or more sweet tastants, indicating direct roles in ligand recognition for all sweet receptors. Receptor response profiles are validated by analysis of taste responses in corresponding Gr mutants. The response matrix shows extensive overlap in Gr-ligand interactions and loosely separates sweet receptors into two groups matching their relationships by sequence. Expression of a bitter taste receptor confers sensitivity to selected aversive tastants that match the responses of the neuron that the Gr is derived from. Finally, an internal fructose-sensing receptor, Gr43a, and its ortholog in the malaria mosquito, AgGr25, were characterized in the ab1C expression system. Both receptors were found to show robust responses to fructose along with a number of other sweet tastants. These results provide a molecular basis for tastant detection by the entire repertoire of sweet taste receptors in the fly and lay the foundation for studying Grs in mosquitoes and other insects that transmit deadly diseases.

Wednesday, February 5th

Cochella, L., Tursun, B., Hsieh, Y. W., Galindo, S., Johnston, R. J., Chuang, C. F. and Hobert, O. (2014). Two distinct types of neuronal asymmetries are controlled by the Caenorhabditis elegans zinc finger transcription factor die-1. Genes Dev 28: 34-43. PubMed ID: 24361693
Summary: Left/right asymmetric features of animals are either randomly distributed on either the left or right side within a population ('antisymmetries') or found stereotypically on one particular side of an animal ('directional asymmetries'). Both types of asymmetries can be found in nervous systems, but whether the regulatory programs that establish these asymmetries share any mechanistic features is not known. This study describes an unprecedented molecular link between these two types of asymmetries in Caenorhabditis elegans. The zinc finger transcription factor die-1 is expressed in a directionally asymmetric manner in the gustatory neuron pair ASE left (ASEL) and ASE right (ASER), while it is expressed in an antisymmetric manner in the olfactory neuron pair AWC left (AWCL) and AWC right (AWCR). Asymmetric die-1 expression is controlled in a fundamentally distinct manner in these two neuron pairs. Importantly, asymmetric die-1 expression controls the directionally asymmetric expression of gustatory receptor proteins in the ASE neurons and the antisymmetric expression of olfactory receptor proteins in the AWC neurons. These asymmetries serve to increase the ability of the animal to discriminate distinct chemosensory inputs.

Kurabayashi, N. and Sanada, K. (2013). Increased dosage of DYRK1A and DSCR1 delays neuronal differentiation in neocortical progenitor cells. Genes Dev 27: 2708-2721. PubMed ID: 24352425
Summary: Down's syndrome (DS), a major genetic cause of mental retardation, arises from triplication of genes on human chromosome 21. This study shows that DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A; Drosophila homolog - Minibrain) and DSCR1 (DS critical region 1; Drosophila homolog - Nebula/Sarah), two genes lying within human chromosome 21 and encoding for a serine/threonine kinase and calcineurin regulator, respectively, are expressed in neural progenitors in the mouse developing neocortex. Increasing the dosage of both proteins in neural progenitors leads to a delay in neuronal differentiation, resulting ultimately in alteration of their laminar fate. This defect is mediated by the cooperative actions of DYRK1A and DSCR1 in suppressing the activity of the transcription factor NFATc. In Ts1Cje mice, a DS mouse model, dysregulation of NFATc in conjunction with increased levels of DYRK1A and DSCR1 was observed. Furthermore, counteracting the dysregulated pathway ameliorates the delayed neuronal differentiation observed in Ts1Cje mice. In sum, these findings suggest that dosage of DYRK1A and DSCR1 is critical for proper neurogenesis through NFATc and provide a potential mechanism to explain the neurodevelopmental defects in DS.

Tsui, D., Voronova, A., Gallagher, D., Kaplan, D. R., Miller, F. D. and Wang, J. (2014). CBP regulates the differentiation of interneurons from ventral forebrain neural precursors during murine development. Dev Biol 385: 230-241. PubMed ID: 24247009
Summary: The mechanisms that regulate appropriate genesis and differentiation of interneurons in the developing mammalian brain are of significant interest not only because interneurons play key roles in the establishment of neural circuitry, but also because when they are deficient, this can cause epilepsy. In this regard, one genetic syndrome that is associated with deficits in neural development and epilepsy is Rubinstein-Taybi Syndrome (RTS), where the transcriptional activator and histone acetyltransferase CBP (see Drosophila Nejire) is mutated and haploinsufficient. This study has asked whether CBP is necessary for the appropriate genesis and differentiation of interneurons in the murine forebrain, since this could provide an explanation for the epilepsy that is associated with RTS. CBP is expressed in neural precursors within the embryonic medial ganglionic eminence (MGE), an area that generates the vast majority of interneurons for the cortex. Using primary cultures of MGE precursors, this study shows that knockdown of CBP causes deficits in differentiation of these precursors into interneurons and oligodendrocytes, and that overexpression of CBP is by itself sufficient to enhance interneuron genesis. Moreover, it was shown that levels of the neurotransmitter synthesis enzyme GAD67, which is expressed in inhibitory interneurons, are decreased in the dorsal and ventral forebrain of neonatal CBP+/- mice, indicating that CBP plays a role in regulating interneuron development in vivo. Thus, CBP normally acts to ensure the differentiation of appropriate numbers of forebrain interneurons, and when its levels are decreased, this causes deficits in interneuron development, providing a potential explanation for the epilepsy seen in individuals with RTS.

Otero, J. J., et al. (2014).. Cerebellar cortical lamination and foliation require cyclin A2. Dev Biol 385: 328-339. PubMed ID: 24184637
Summary: The mammalian genome encodes two A-type cyclins (see Drosophila Cyclin A), which are considered potentially redundant yet essential regulators of the cell cycle. This study tested requirements for cyclin A1 and cyclin A2 function in cerebellar development. Compound conditional loss of cyclin A1/A2 in neural progenitors resulted in severe cerebellar hypoplasia, decreased proliferation of cerebellar granule neuron progenitors (CGNP), and Purkinje (PC) neuron dyslamination. Deletion of cyclin A2 alone showed an identical phenotype, demonstrating that cyclin A1 does not compensate for cyclin A2 loss in neural progenitors. Cyclin A2 loss lead to increased apoptosis at early embryonic time points but not at post-natal time points. In contrast, neural progenitors of the VZ/SVZ did not undergo increased apoptosis, indicating that VZ/SVZ-derived and rhombic lip-derived progenitor cells show differential requirements to cyclin A2. Conditional knockout of cyclin A2 or the SHH proliferative target Nmyc in CGNP also resulted in PC neuron dyslamination. Although cyclin E1 has been reported to compensate for cyclin A2 function in fibroblasts and is upregulated in cyclin A2 null cerebella, cyclin E1 expression was unable to compensate for loss-of cyclin A2 function.

Tuesday, February 4th

Hayward, D., Metz, J., Pellacani, C. and Wakefield, J. G. (2014). Synergy between Multiple Microtubule-Generating Pathways Confers Robustness to Centrosome-Driven Mitotic Spindle Formation. Dev Cell 28: 81-93. PubMed ID: 24389063
Summary: The mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromosome alignment and segregation. Although different cells have been shown to use different molecular pathways to generate the microtubules required for spindle formation, how these pathways are coordinated within a single cell is poorly understood. This study has tested the limits within which the Drosophila embryonic spindle forms, disrupting the inherent temporal control that overlays mitotic microtubule generation, interfering with the molecular mechanism that generates new microtubules from preexisting ones, and disrupting the spatial relationship between microtubule nucleation and the usually dominant centrosome. This work uncovers the possible routes to spindle formation in embryos and establishes the central role of Augmin (an eight-subunit complex that increases the number of spindle MTs, apparently by binding to preexisting MTs and recruiting γ-TuRC), in all microtubule-generating pathways. It also demonstrates that the contributions of each pathway to spindle formation are integrated, highlighting the remarkable flexibility with which cells can respond to perturbations that limit their capacity to generate microtubules. Therefore clear evidence has been provided that all three pathways to spindle formation (centrosomal, chromatin, and acentrosomal MT organizing center driven are dependent on a fourth: Augmin. Together with recent work demonstrating that new MTs can be produced in an Augmin-dependent manner using preexisting ones generated in vitro in Xenopus egg extracts, this evidence suggests that this conserved protein complex, once active, works on all existing mitotic MTs.

Jayanandanan, N., Mathew, R. and Leptin, M. (2014). Guidance of subcellular tubulogenesis by actin under the control of a synaptotagmin-like protein and Moesin. Nat Commun 5: 3036. PubMed ID: 24413568
Summary: Apical membranes in many polarized epithelial cells show specialized morphological adaptations that fulfil distinct physiological functions. The air-transporting tubules of Drosophila tracheal terminal cells represent an extreme case of membrane specialization. This study shows that Bitesize (Btsz), a synaptotagmin-like protein family member, is needed for luminal membrane morphogenesis. Unlike in multicellular tubes and other epithelia, where it influences apical integrity by affecting adherens junctions, Btsz here acts at a distance from junctions. Localized at the luminal membrane through its tandem C2 domain, it recruits activated Moesin. Both proteins are needed for the integrity of the actin cytoskeleton at the luminal membrane, but not for other pools of F-actin in the cell, nor do actin-dependent processes at the outer membrane, such as filopodial activity or membrane growth depend on Btsz. Btsz and Moesin guide luminal membrane morphogenesis through organizing actin and allowing the incorporation of membrane containing the apical determinant Crumbs.

Chen, Y., Rolls, M. M. and Hancock, W. O. (2014). An EB1-Kinesin Complex Is Sufficient to Steer Microtubule Growth In Vitro. Curr. Biol. 24: 316-321. PubMed ID: 24462004
Summary: Proper microtubule polarity underlies overall neuronal polarity, but mechanisms for maintaining microtubule polarity are not well understood. Previous live imaging in Drosophila dendritic arborization neurons showed that while microtubules are uniformly plus-end out in axons, dendrites possess uniformly minus-end-out microtubules. Thus, maintaining uniform microtubule polarity in dendrites requires that growing microtubule plus ends entering branch points be actively directed toward the cell body. A model has been proposed in which EB1 tracks the plus ends of microtubules growing into a branch and an associated kinesin-2 motor walks along a static microtubule to steer the plus end toward the cell body. However, the fast plus-end binding dynamics of EB1 appear to be at odds with this proposed mechanical function. To test this model in vitro, the system was reconstituted by artificially dimerizing EB1 to kinesin, growing microtubules from immobilized seeds, and imaging encounters between growing microtubule plus ends and static microtubules. Consistent with in vivo observations, the EB1-kinesin complex actively steered growing microtubules. Thus, EB1 kinetics and mechanics are sufficient to bend microtubules for several seconds. Other kinesins also demonstrated this activity, suggesting this is a general mechanism for organizing and maintaining proper microtubule polarity in cells.

Doodhi, H., Katrukha, E. A., Kapitein, L. C. and Akhmanova, A. (2014). Mechanical and Geometrical Constraints Control Kinesin-Based Microtubule Guidance. Curr Biol. 24: 322-328. PubMed ID: 24462000
Summary: Proper organization of microtubule networks depends on microtubule-associated proteins and motors that use different spatial cues to guide microtubule growth. For example, it has been proposed that the uniform minus-end-out microtubule organization in dendrites of Drosophila neurons is maintained by steering of polymerizing microtubules along the stable ones by kinesin-2 motors bound to growing microtubule plus ends. To explore the mechanics of kinesin-guided microtubule growth, this process was reconstituted in vitro. In the presence of microtubule plus-end tracking EB proteins, a constitutively active kinesin linked to the EB-interacting motif SxIP effectively guided polymerizing microtubules along other microtubules both in cells and in vitro. Experiments combined with modeling revealed that at angles larger than 90°, guidance efficiency is determined by the force needed for microtubule bending. At angles smaller than 90°, guidance requires microtubule growth, and guidance efficiency depends on the ability of kinesins to maintain contact between the two microtubules despite the geometrical constraints imposed by microtubule length and growth rate. These findings provide a conceptual framework for understanding microtubule guidance during the generation of different types of microtubule arrays.

Monday, February 3rd

Hudish, L. I., Blasky, A. J. and Appel, B. (2013). miR-219 regulates neural precursor differentiation by direct inhibition of apical par polarity proteins. Dev Cell 27: 387-398. PubMed ID: 24239515
Summary: Asymmetric self-renewing division of neural precursors is essential for brain development. Partitioning-defective (Par) proteins promote self-renewal, and their asymmetric distribution provides a mechanism for asymmetric division. Near the end of neural development, most asymmetric division ends and precursors differentiate. This correlates with Par protein disappearance, but mechanisms that cause downregulation are unknown. MicroRNAs can promote precursor differentiation but have not been linked to Par protein regulation. This study tested a hypothesis that microRNA miR-219 promotes precursor differentiation by inhibiting Par proteins. Neural precursors in zebrafish larvae lacking miR-219 function retained apical proteins, remained in the cell cycle, and failed to differentiate. miR-219 inhibited expression via target sites within the 3' untranslated sequence of pard3 (see Drosophila Bazooka) and prkci(see Drosophila atypical protein kinase C) mRNAs, which encode Par proteins, and blocking miR-219 access to these sites phenocopied loss of miR-219 function. It is proposed that negative regulation of Par protein expression by miR-219 promotes cell-cycle exit and differentiation.

Hoffmann, S. A., Hos, D., Kuspert, M., Lang, R. A., Lovell-Badge, R., Wegner, M. and Reiprich, S. (2014). Stem cell factor Sox2 and its close relative Sox3 have differentiation functions in oligodendrocytes. Development 141: 39-50. PubMed ID: 24257626
Summary: Neural precursor cells of the ventricular zone give rise to all neurons and glia of the central nervous system and rely for maintenance of their precursor characteristics on the closely related SoxB1 transcription factors Sox1, Sox2 and SoxB3 (see Drosophila Dichaete). This study shows in mouse spinal cord that, whereas SoxB1 proteins are usually downregulated upon neuronal specification, they continue to be expressed in glial precursors. In the oligodendrocyte lineage, Sox2 and Sox3 remain present into the early phases of terminal differentiation. Surprisingly, their deletion does not alter precursor characteristics but interferes with proper differentiation. Although a direct influence on myelin gene expression may be part of their function, evidence is provided for another mode of action. SoxB1 proteins promote oligodendrocyte differentiation in part by negatively controlling miR145 and thereby preventing this microRNA from inhibiting several pro-differentiation factors. This study presents one of the few cases in which SoxB1 proteins, including the stem cell factor Sox2, are associated with differentiation rather than precursor functions.

Manavalan, M. A., Gaziova, I. and Bhat, K. M. (2013). The Midline Protein Regulates Axon Guidance by Blocking the Reiteration of Neuroblast Rows within the Drosophila Ventral Nerve Cord. PLoS Genet 9: e1004050. PubMed ID: 24385932
Summary: Guiding axon growth cones towards their targets is a fundamental process that occurs in a developing nervous system. Several major signaling systems are involved in axon-guidance, and disruption of these systems causes axon-guidance defects. However, the specific role of the environment in which axons navigate in regulating axon-guidance has not been examined in detail. In Drosophila, the ventral nerve cord is divided into segments, and half-segments and the precursor neuroblasts are formed in rows and columns in individual half-segments. The row-wise expression of segment-polarity genes within the neuroectoderm provides the initial row-wise identity to neuroblasts. This study shows that in embryos mutant for the gene midline, which encodes a T-box DNA binding protein, row-2 neuroblasts and their neuroectoderm adopt a row-5 identity. This reiteration of row-5 ultimately creates a non-permissive zone or a barrier, which prevents the extension of interneuronal longitudinal tracts along their normal anterior-posterior path. While the nature of the barrier is not know, the axon tracts either stall when they reach this region or project across the midline or towards the periphery along this zone. Previously studies have shown that midline ensures ancestry-dependent fate specification in a neuronal lineage. These results provide the molecular basis for the axon guidance defects in midline mutants and the significance of proper specification of the environment to axon-guidance. These results also reveal the importance of segmental polarity in guiding axons from one segment to the next, and a link between establishment of broad segmental identity and axon guidance.

Long, S. K., Fulkerson, E., Breese, R., Hernandez, G., Davis, C., Melton, M. A., Chandran, R. R., Butler, N., Jiang, L. and Estes, P. (2014). A Comparison of Midline and Tracheal Gene Regulation during Drosophila Development. PLoS One 9: e85518. PubMed ID: 24465586
Summary: Within the Drosophila embryo, two related bHLH-PAS proteins, Single-minded and Trachealess, control development of the central nervous system midline and the trachea, respectively. These two proteins are bHLH-PAS transcription factors and independently form heterodimers with another bHLH-PAS protein, Tango. During early embryogenesis, expression of Single-minded is restricted to the midline and Trachealess to the trachea and salivary glands, whereas Tango is ubiquitously expressed. Both Single-minded/Tango and Trachealess/Tango heterodimers bind to the same DNA sequence, called the CNS midline element (CME) within cis-regulatory sequences of downstream target genes. While Single-minded/Tango and Trachealess/Tango activate some of the same genes in their respective tissues during embryogenesis, they also activate a number of different genes restricted to only certain tissues. The goal of this research is to understand how these two related heterodimers bind different enhancers to activate different genes, thereby regulating the development of functionally diverse tissues. Existing data indicates that Single-minded and Trachealess may bind to different co-factors restricted to various tissues, causing them to interact with the CME only within certain sequence contexts. This would lead to the activation of different target genes in different cell types. To understand how the context surrounding the CME is recognized by different bHLH-PAS heterodimers and their co-factors, novel enhancers were identified and analyzed that drive midline and/or tracheal expression, and they were compared to previously characterized enhancers. In addition, expression was tested of synthetic reporter genes containing the CME flanked by different sequences. Taken together, these experiments identify elements overrepresented within midline and tracheal enhancers and suggest that sequences immediately surrounding a CME help dictate whether a gene is expressed in the midline or trachea.

Sunday, February 2nd

Rembold, M., Ciglar, L., Yanez-Cuna, J. O., Zinzen, R. P., Girardot, C., Jain, A., Welte, M. A., Stark, A., Leptin, M. and Furlong, E. E. (2014). A conserved role for Snail as a potentiator of active transcription. Genes Dev 28: 167-181. PubMed ID: 24402316
Summary: The transcription factors of the Snail family are key regulators of epithelial-mesenchymal transitions, cell morphogenesis, and tumor metastasis. Since its discovery in Drosophila approximately 25 years ago, Snail has been extensively studied for its role as a transcriptional repressor. This study demonstrate that Drosophila Snail can positively modulate transcriptional activation. By combining information on in vivo occupancy with expression profiling of hand-selected, staged snail mutant embryos, 106 genes were identified that are potentially directly regulated by Snail during mesoderm development. In addition to the expected Snail-repressed genes, almost 50% of Snail targets showed an unanticipated activation. The majority of 'Snail-activated' genes have enhancer elements cobound by Twist and are expressed in the mesoderm at the stages of Snail occupancy. Snail can potentiate Twist-mediated enhancer activation in vitro and is essential for enhancer activity in vivo. Using a machine learning approach, it was shown that differentially enriched motifs are sufficient to predict Snail's regulatory response. In silico mutagenesis revealed a likely causative motif, which this study demonstrates to be essential for enhancer activation. Taken together, these data indicate that Snail can potentiate enhancer activation by collaborating with different activators, providing a new mechanism by which Snail regulates development.

Buckley, M. S., Kwak, H., Zipfel, W. R. and Lis, J. T. (2014). Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation. Genes Dev 28: 14-19. PubMed ID: 24395245
Summary: The kinetics with which promoter-proximal paused RNA polymerase II (Pol II) undergoes premature termination versus productive elongation is central to understanding underlying mechanisms of metazoan transcription regulation. To assess the fate of Pol II quantitatively, photoactivatable GFP-tagged Pol II at uninduced Hsp70 was tracked on polytene chromosomes, and it was shown that Pol II is stably paused with a half-life of 5 min. Biochemical analysis of short nascent RNA from Hsp70 reveals that this half-life is determined by two comparable rates of productive elongation and premature termination of paused Pol II. Importantly, heat shock dramatically increases elongating Pol II without decreasing termination, indicating that regulation acts at the step of paused Pol II entry to productive elongation.

Menet, J. S., Pescatore, S. and Rosbash, M. (2014). CLOCK:BMAL1 is a pioneer-like transcription factor. Genes Dev 28: 8-13. PubMed ID: 24395244
Summary: The mammalian circadian clock relies on the master genes CLOCK and BMAL1 to drive rhythmic gene expression and regulate biological functions under circadian control. This study shows that rhythmic CLOCK:BMAL1 (see Drosophila Clock and Cycle) DNA binding promotes rhythmic chromatin opening. Mechanisms include CLOCK:BMAL1 binding to nucleosomes and rhythmic chromatin modification; e.g., incorporation of the histone variant H2A.Z. This rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLOCK:BMAL1, suggesting that the activity of these other transcription factors contributes to the genome-wide CLOCK:BMAL1 heterogeneous transcriptional output. These data therefore indicate that the clock regulation of transcription relies on the rhythmic regulation of chromatin accessibility and suggest that the concept of pioneer function extends to acute gene regulation.

Saturday, February 1st

Erceg, J., Saunders, T. E., Girardot, C., Devos, D. P., Hufnagel, L. and Furlong, E. E. (2014). Subtle Changes in Motif Positioning Cause Tissue-Specific Effects on Robustness of an Enhancer's Activity. PLoS Genet 10: e1004060. PubMed ID: 24391522
Summary: Deciphering the specific contribution of individual motifs within cis-regulatory modules (CRMs) is crucial to understanding how gene expression is regulated and how this process is affected by sequence variation. But despite vast improvements in the ability to identify where transcription factors (TFs) bind throughout the genome, the ability to relate information on motif occupancy to function from sequence alone is limited. This study engineered 63 synthetic CRMs to systematically assess the relationship between variation in the content and spacing of motifs within CRMs to CRM activity during development using Drosophila transgenic embryos. In over half the cases, very simple elements containing only one or two types of TF binding motifs were capable of driving specific spatio-temporal patterns during development. Different motif organizations provide different degrees of robustness to enhancer activity, ranging from binary on-off responses to more subtle effects including embryo-to-embryo and within-embryo variation. By quantifying the effects of subtle changes in motif organization, it was possible to model biophysical rules that explain CRM behavior and may contribute to the spatial positioning of CRM activity in vivo. For the same enhancer, the effects of small differences in motif positions varied in developmentally related tissues, suggesting that gene expression may be more susceptible to sequence variation in one tissue compared to another. This result has important implications for human eQTL studies in which many associated mutations are found in cis-regulatory regions, though the mechanism for how they affect tissue-specific gene expression is often not understood.

Martinez, C., Rest, J. S., Kim, A. R., Ludwig, M., Kreitman, M., White, K. and Reinitz, J. (2014). Ancestral resurrection of the Drosophila S2E enhancer reveals accessible evolutionary paths through compensatory change. Mol Biol Evol. PubMed ID: 24408913
Summary: Upstream regulatory sequences that control gene expression evolve rapidly, yet the expression patterns and functions of most genes are typically conserved. In order to address this paradox, this study has reconstructed computationally and resurrected in vivo the cis-regulatory regions of the ancestral Drosophila eve stripe 2 element and evaluated its evolution using a mathematical model of promoter function. A feed-forward transcriptional model predicts gene expression patterns directly from enhancer sequence. This functional model was used along with phylogenetics to generate a set of possible ancestral eve stripe 2 sequences for the common ancestors of 1) Drosophila simulans and D. sechellia, 2) D. melanogaster, D. simulans, D. sechellia, and 3) D. erecta and D. yakuba. These ancestral sequences were synthesized and resurrected in vivo. Using a combination of quantitative and computational analysis, clear support was foumd for functional compensation between the binding sites for Bicoid, Giant, and Kruppel over the course of 40-60 million years of Drosophila evolution. This compensation is driven by a coupling interaction between Bicoid activation and repression at the anterior and posterior border necessary for proper placement of the anterior stripe 2 border. A multiplicity of mechanisms for binding site turnover exemplified by Bicoid, Giant, and Kruppel sites, explains how rapid sequence change may occur while maintaining the function of the cis-regulatory element.

Ming, L., Wilk, R., Reed, B. H. and Lipshitz, H. D. (2014). Drosophila Hindsight and mammalian RREB-1 are evolutionarily conserved DNA-binding transcriptional attenuators. Differentiation. PubMed ID: 24418439
Summary: The Drosophila Hindsight (hnt) gene encodes a C2H2-type Zinc-finger protein, HNT, that plays multiple developmental roles including control of embryonic germ band retraction and regulation of retinal cell fate and morphogenesis. While the developmental functions of the human HNT homolog, RREB-1, are unknown, it has been shown to function as a transcriptional modulator of several tumor suppressor genes. This study investigated HNT's functional motifs, target genes and its regulatory abilities. The C-terminal region of HNT, containing the last five of its 14 Zinc fingers, binds in vitro to DNA elements very similar to those identified for RREB-1. HNT's in vivo binding sites were mapped on salivary gland polytene chromosomes, and where HNT is bound to two target genes, hnt itself and nervy (nvy) was defined at high resolution. Data from both loss-of-function and over-expression experiments show that HNT attenuates the transcription of these two targets in a tissue-specific manner. RREB-1, when expressed in Drosophila, binds to the same polytene chromosome sites as HNT, attenuates expression of the hnt and nvy genes, and rescues the germ band retraction phenotype. HNT's ninth Zinc finger has degenerated or been lost in the vertebrate lineage. A HNT protein mutant for this finger can also attenuate target gene expression and rescue germ band retraction. Thus HNT and RREB-1 are functional homologs at the level of DNA binding, transcriptional regulation and developmental control.

McKay, D. J. and Lieb, J. D. (2013). A common set of DNA regulatory elements shapes Drosophila appendages. Dev Cell 27: 306-318. PubMed ID: 24229644
Summary: Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different 'master regulator' transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. This study used genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. It is proposed that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules.

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